ARABINOSE ISOMERASES FOR YEAST

Abstract

A group of arabinose isomerases are disclosed that provide effective amounts of activity for use of arabinose in production of ethanol, when expressed in yeast cells expressing the other enzymes of an arabinose utilization pathway. The group of arabinose isomerases represents a clade of a phylogenetic tree, having a distinguishing conserved amino acid sequence motif. Other useful arabinose isomerases are also disclosed.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 62/319,945 (filed Apr. 8, 2016), which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[0002] The field of invention relates to genetic engineering, and more specifically to engineering yeast to enhance utilization of arabinose during fermentation.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named CL6357WOPCT_SequenceListing_ST25_ExtraLinesRemoved created on Mar. 31, 2017, and having a size of 284 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND

[0004] Currently, fermentative production of ethanol is typically done with yeast, particularly Saccharomyces cerevisiae, using hexoses obtained from grains or mash as the carbohydrate source. Use of hydrolysate prepared from cellulosic biomass as a carbohydrate source for fermentation is desirable, as this is a readily renewable resource that does not compete with the food supply. The most abundant sugar in cellulosic biomass hydrolysate is glucose, while the pentoses xylose and arabinose are also present. Many biocatalysts, including the yeast Saccharomyces cerevisiae, are not naturally capable of metabolizing xylose or arabinose, but can be engineered to express xylose and/or arabinose utilization pathways. One approach to engineering a xylose utilization pathway in yeast includes introduction of xylose isomerase, and increasing expression of the pentose phosphate pathway including xylulokinase, transaldolase, transketolase 1, D-ribulose-5-phosphate 3-epimerase, and ribose 5-phosphate ketol-isomerase. Use of arabinose can be achieved by additionally engineering expression of L-arabinose isomerase, L-ribulokinase, and L-ribulose-5-phosphate 4-epimerase (e.g., Becker and Boles, Appl. Environ. Microbiol. 69:4144-4150).

[0005] Though yeast strains engineered in this manner can utilize arabinose, such arabinose use is typically inefficient due to poor efficiency of arabinose isomerase, which operates at the first step of the bacterial-type arabinose assimilation pathway. Thus, there remains a need for arabinose isomerases that are more effective when expressed in yeast, and engineered yeast cells that express an arabinose isomerase that allows greater efficiency of arabinose utilization.

SUMMARY

[0006] In one embodiment, the present disclosure concerns a recombinant yeast cell comprising an arabinose utilization pathway that comprises a polypeptide having arabinose isomerase activity, wherein the yeast cell comprises a heterologous polynucleotide encoding the polypeptide, wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:67, and wherein the position of the motif in the polypeptide corresponds with positions 237-269 of SEQ ID NO:7.

[0007] In another embodiment, the present disclosure concerns a method for producing a yeast cell having arabinose isomerase activity. This method comprises: (a) providing a yeast cell lacking arabinose isomerase; and (b) introducing a heterologous polynucleotide into the yeast cell, wherein the heterologous polynucleotide encodes a polypeptide having arabinose isomerase activity, and wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:67.

[0008] In another embodiment, the present disclosure concerns a method of producing a target compound from arabinose comprising: (a) providing a recombinant yeast cell as disclosed herein; (b) growing the yeast cell of (a) in medium comprising arabinose, wherein the target compound is produced; and (c) optionally isolating the target compound of (b).

[0009] In another embodiment, the present disclosure concerns a recombinant yeast cell comprising an arabinose utilization pathway that comprises a polypeptide having arabinose isomerase activity, wherein the yeast cell comprises a heterologous polynucleotide encoding the polypeptide, and wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:15 or 20. The present disclosure also concerns a method of using such a yeast cell to produce a target compound from arabinose comprising: (a) providing the yeast cell; (b) growing the yeast cell of (a) in medium comprising arabinose, wherein the target compound is produced; and c) optionally isolating the target compound of (b).

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

[0010] FIG. 1 shows a phylogenetic tree of twenty candidate arabinose isomerase proteins, and the B. subtilis arabinose isomerase protein.

[0011] FIG. 2A shows the 237-269 Motif formula I (SEQ ID NO:67).

[0012] FIG. 2B shows the 237-269 Motif formula II (SEQ ID NO:67) with bold and underline highlighting of specific positions, as described in the detailed description.

[0013] FIG. 2C shows an alignment of twenty candidate arabinose isomerases and the B. subtilis arabinose isomerase in the region of positions 237-269 (with reference to SEQ ID NO:7), with the six members of the 237-269 Motif clade at the top. The BSaraA amino acid sequence used in this alignment is SEQ ID NO:41.

[0014] FIG. 3A is a plasmid map of pSX01 (SEQ ID NO:43).

[0015] FIG. 3B is a plasmid map of pSX208 (SEQ ID NO:47).

[0016] FIG. 4A is a plasmid map of pSX209 (SEQ ID NO:48).

[0017] FIG. 4B is a plasmid map of pSX210 (SEQ ID NO:49).

[0018] FIG. 5A is a plasmid map of pSA0-B (SEQ ID NO:58).

[0019] FIG. 5B is a plasmid map of pSA503 (SEQ ID NO:59).

[0020] FIG. 6 is a graph showing growth (OD600), arabinose use, and production of ethanol during fermentation by yeast strain PX182-araBAD5030 in medium containing arabinose as the only sugar.

[0021] FIG. 7 is a graph of in vitro arabinose isomerase activities (.mu.mol/mg/min) from arabinose- and xylose-utilizing yeast strains expressing different candidate arabinose isomerases or the B. subtilis arabinose isomerase.

TABLE-US-00001

[0022] TABLE 1 SEQ ID NOs for Amino Acid (AA) and Nucleotide (NT) Sequences (Codon-Optimized Coding Regions) of Candidate Arabinose Isomerases. Designation SEQ ID NO: AA SEQ ID NO: NT HMPREF9412_4417 1 21 POTG_01507 2 22 HMPREF9374_3716 3 23 DORFOR_01282 4 24 HMPREF0994_04908 5 25 NODE_4061684 6 26 NODE_3664377 7 27 NODE_458803 8 28 NODE_3921064 9 29 NODE_3693095 10 30 DORLON_00938 11 31 HMPREF9469_04726 12 32 HMPREF9467_00216 13 33 RTO_26010 14 34 BRYFOR_08166 15 35 RUMOBE_03031 16 36 NODE_3658038 17 37 NODE_4175755 18 38 NODE_2588280 19 39 NODE_3735508 20 40

[0023] Arabinose isomerase designations in Table 1 are from the cow rumen metagenome dataset ("NODE" designations; Hess et al., Science 331:463-467, incorporated herein by reference) or the human microbiome dataset (The Human Microbiome Jumpstart Reference Strains Consortium et al., Science 328:994-999, incorporated herein by reference).

[0024] SEQ ID NO:41 is the amino acid sequence of the arabinose isomerase from B. subtilis (BSaraA).

[0025] SEQ ID NO:42 is the nucleotide sequence of the codon-optimized coding to region for the B. subtilis arabinose isomerase.

[0026] SEQ ID NO:43 is the nucleotide sequence of the plasmid pSX01.

[0027] SEQ ID NO:44 is the amino acid sequence of the xylose isomerase VDxylA.

[0028] SEQ ID NO:45 is the nucleotide sequence of the codon-optimized coding region for the VDxylA arabinose isomerase.

[0029] SEQ ID NO:46 is the nucleotide sequence of the 2966-bp chimeric expression cassette designated as UAS(FBA1)::PDC1p::VDxylA::ILV5t.

[0030] SEQ ID NO:47 is the nucleotide sequence of the plasmid pSX208.

[0031] SEQ ID NO:48 is the nucleotide sequence of the plasmid pSX209.

[0032] SEQ ID NO:49 is the nucleotide sequence of the plasmid pSX210.

[0033] SEQ ID NO:50 is the nucleotide sequence of the CRE recombinase vector pJT254.

[0034] SEQ ID NO:51 is the nucleotide sequence of the 2429-bp chimeric expression cassette designated as ADHp::BSaraA::CYC1t.

[0035] SEQ ID NO:52 is the amino acid sequence of the E. coli araB gene-encoded ribulokinase.

[0036] SEQ ID NO:53 is the nucleotide sequence of the codon-optimized coding region for the E. coli ribulokinase.

[0037] SEQ ID NO:54 is the nucleotide sequence of the 2907-bp chimeric expression cassette designated as ILV5p::ECaraB::PHO13-3'UTR.

[0038] SEQ ID NO:55 is the amino acid sequence of the E. coli araD gene-encoded L-ribulose-5-phosphate 4-epimerase.

[0039] SEQ ID NO:56 is the nucleotide sequence of the codon-optimized coding region for the E. coli L-ribulose-5-phosphate 4-epimerase.

[0040] SEQ ID NO:57 is the nucleotide sequence of the 1691-bp chimeric expression cassette designated as GPDp::ECaraD::ADH1t.

[0041] SEQ ID NO:58 is the nucleotide sequence of the plasmid pSA0-B.

[0042] SEQ ID NO:59 is the nucleotide sequence of the plasmid pSA503.

[0043] SEQ ID NO:60 is the amino acid sequence of the arabinose isomerase from E. coli.

[0044] SEQ ID NO:61 is the amino acid sequence of the arabinose isomerase from Bacillus licheniformis.

[0045] SEQ ID NO:62 is the amino acid sequence of the arabinose isomerase from Clostridium acetobutylicum.

[0046] SEQ ID NO:63 is the amino acid sequence of the arabinose isomerase from Leuconostoc mesenteroides.

[0047] SEQ ID NO:64 is the amino acid sequence of the arabinose isomerase from Lactobacillus plantarum.

[0048] SEQ ID NO:65 is the amino acid sequence of the arabinose isomerase from Pediococcus pentosaceus.

[0049] SEQ ID NO:66 is the amino acid sequence representing a "237-269 Motif".

[0050] SEQ ID NO:67 is the amino acid sequence of the 237-269 Motif shown in FIG. 2A.

[0051] Each of SEQ ID NOs:68-71 is an amino acid sequence that can optionally be excluded in certain embodiments of the present disclosure.

DETAILED DESCRIPTION

[0052] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0053] Unless otherwise disclosed, the terms "a" and "an" as used herein are intended to encompass one or more (i.e., at least one) of a stated feature.

[0054] Where present, all ranges are inclusive and combinable, except as otherwise noted. For example, when a range of "1 to 5" is recited, the recited range should be construed as including ranges "1 to 4", "1 to 3", "1-2", "1-2 & 4-5", "1-3 & 5", and the like.

[0055] The terms "about", "approximately" and the like in some aspects, as used to modify certain numerical values herein, refer to being within 5%-10% of the stated numerical value.

[0056] The terms "arabinose isomerase", "L-arabinose isomerase" and the like refer to an enzyme that catalyzes the conversion of L-arabinose to L-ribulose. Arabinose isomerases belong to the group of enzymes classified in Enzyme Commission (EC) entry 5.3.1.4.

[0057] The terms "carbon substrate", "fermentable carbon substrate" and the like refer to a carbon source capable of being metabolized by microorganisms. A type of carbon substrate is "fermentable sugars" which refer to oligosaccharides and monosaccharides that can be used as a carbon source by a microorganism in a fermentation process. Arabinose and xylose are examples of fermentable sugars.

[0058] The term "lignocellulosic" refers to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose.

[0059] The term "cellulosic" refers to a composition comprising cellulose and additional components, which may include hemicellulose and lignin.

[0060] The term "saccharification" refers to the production of fermentable sugars from polysaccharides such as cellulose and hemicellulose. Saccharification can be done via chemical and/or enzymatic means.

[0061] "Biomass" refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Further examples of biomass include, but are not limited to, corn cobs, crop residues such as corn husks, corn stover, corn grain fiber, grasses, beet pulp, wheat straw, wheat chaff, oat straw, barley straw, barley hulls, hay, rice straw, rice hulls, switchgrass, miscanthus, cord grass, reed canary grass, waste paper, sugar cane bagasse, sorghum bagasse, sorghum stover, soybean stover, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, palm waste, shrubs and bushes, vegetables, fruits, flowers, and animal manure.

[0062] The term "pretreated biomass" refers to biomass that has been subjected to thermal, physical and/or chemical treatment to increase the availability of polysaccharides in the biomass to saccharification enzymes. Biomass pretreatment is typically done before saccharification.

[0063] "Biomass hydrolysate" refers to the product resulting from saccharification of biomass, and comprises fermentable sugars.

[0064] The terms "target compound", "target chemical" and the like refer to a compound made by a microorganism via an endogenous or recombinant biosynthetic/metabolic pathway that is able to metabolize a fermentable carbon source to produce the target compound.

[0065] The terms "percent by volume", "volume percent", "vol %", "v/v %" and the like are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)].times.100%.

[0066] The terms "percent by weight", "weight percentage (wt %)", "weight-weight percentage (% w/w)" and the like are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.

[0067] The terms "polynucleotide", "polynucleotide sequence", "nucleic acid molecule" and the like are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (ribonucleotides or deoxyribonucleotides) can be referred to by a single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate (for RNA or DNA, respectively), "G" for guanylate or deoxyguanylate (for RNA or DNA, respectively), "U" for uridylate (for RNA), "T" for deoxythymidylate (for DNA), "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, "W" for A or T, and "N" for any nucleotide (e.g., N can be A, C, T, or G, if referring to a DNA sequence; N can be A, C, U, or G, if referring to an RNA sequence).

[0068] The terms "motif", "conserved motif" and the like herein refer to a distinctive and recurring structural unit, such as within an amino acid sequence. By "recurring" it is meant that a motif occurs in multiple related polypeptides, for example.

[0069] Herein, a first polynucleotide sequence that is "complementary" to a second polynucleotide sequence can alternatively be referred to as being in the "antisense" orientation with the second sequence.

[0070] The term "gene" as used herein refers to a DNA polynucleotide sequence that expresses an RNA (RNA is transcribed from the DNA polynucleotide sequence) from a coding region, which RNA can be a messenger RNA (encoding a protein) or a non-protein-coding RNA. A gene may refer to the coding region alone, or may include regulatory sequences upstream and/or downstream to the coding region (e.g., promoters, 5'-untranslated regions, 3'-transcription terminator regions). A coding region encoding a protein can alternatively be referred to herein as an "open reading frame" (ORF). A gene that is "native" or "endogenous" refers to a gene as found in nature with its own regulatory sequences; such a gene is located in its natural location in the genome of a host cell. A "chimeric" gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature (i.e., the regulatory and coding regions are heterologous with each other). Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A "foreign" or "heterologous" gene can refer to a gene that is introduced into the host organism by gene transfer. Foreign/heterologous genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. The polynucleotide sequences in certain embodiments disclosed herein are heterologous. A "transgene" is a gene that has been introduced into the genome by a gene delivery procedure (e.g., transformation). A "codon-optimized" open reading frame has its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

[0071] The term "heterologous" means not naturally found in the location of interest. For example, a heterologous gene can be one that is not naturally found in a host organism, but that is introduced into the host organism by gene transfer. As another example, a nucleic acid molecule that is present in a chimeric gene can be characterized as being heterologous, as such a nucleic acid molecule is not naturally associated with the other segments of the chimeric gene (e.g., a promoter can be heterologous to a coding sequence).

[0072] A "non-native" amino acid sequence or polynucleotide sequence comprised in a cell or organism herein does not occur in a native (natural) counterpart of such cell or organism. Such an amino acid sequence or polynucleotide sequence can also be referred to as being heterologous to the cell or organism.

[0073] "Regulatory sequences" as used herein refer to nucleotide sequences located upstream of a gene's transcription start site (e.g., promoter), 5' untranslated regions, introns, and 3' non-coding regions, and which may influence the transcription, processing or stability, and/or translation of an RNA transcribed from the gene. Regulatory sequences herein may include promoters, enhancers, silencers, 5' untranslated leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, and other elements involved in regulation of gene expression. One or more regulatory elements herein may be heterologous to a coding region herein.

[0074] A "promoter" as used herein refers to a DNA sequence capable of controlling the transcription of RNA from a gene. In general, a promoter sequence is upstream of the transcription start site of a gene. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Promoters that cause a gene to be expressed in a cell at most times under all circumstances are commonly referred to as "constitutive promoters". One or more promoters herein may be heterologous to a coding region herein.

[0075] A "strong promoter" as used herein refers to a promoter that can direct a relatively large number of productive initiations per unit time, and/or is a promoter driving a higher level of gene transcription than the average transcription level of the genes in a cell.

[0076] The terms "3' non-coding sequence", "transcription terminator" and "terminator" as used herein refer to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.

[0077] The terms "upstream" and "downstream" as used herein with respect to polynucleotides refer to "5' of" and "3' of", respectively.

[0078] The term "expression" as used herein refers to (i) transcription of RNA (e.g., mRNA or a non-protein-coding RNA) from a coding region, and/or (ii) translation of a polypeptide from mRNA. Expression of a coding region of a polynucleotide sequence can be up-regulated or down-regulated in certain embodiments.

[0079] The term "operably linked" as used herein refers to the association of two or more nucleic acid sequences such that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence. That is, the coding sequence is under the transcriptional control of the promoter. A coding sequence can be operably linked to one (e.g., promoter) or more (e.g., promoter and terminator) regulatory sequences, for example.

[0080] The term "recombinant" when used herein to characterize a DNA sequence such as a plasmid, vector, or construct refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis and/or by manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0081] The term "transformation" as used herein refers to the transfer of a nucleic acid molecule into a host organism or host cell by any method. A nucleic acid molecule that has been transformed into an organism/cell may be one that replicates autonomously in the organism/cell, or that integrates into the genome of the organism/cell, or that exists transiently in the cell without replicating or integrating. Non-limiting examples of nucleic acid molecules suitable for transformation are disclosed herein, such as plasmids and linear DNA molecules. Host organisms/cells herein containing a transforming nucleic acid sequence can be referred to as "transgenic", "recombinant", "transformed", "engineered", as a "transformant", and/or as being "modified for exogenous gene expression", for example.

[0082] The terms "control cell" and "suitable control cell" are used interchangeably herein and may be referenced with respect to a cell in which a particular modification (e.g., over-expression of a polynucleotide, down-regulation of a polynucleotide) has been made (i.e., an "experimental cell"). A control cell may be any cell that does not have or does not express the particular modification of the experimental cell. Thus, a control cell may be an untransformed wild type cell or may be genetically transformed but does not express the particular modification. For example, a control cell may be a direct parent of the experimental cell, which direct parent cell does not have the particular modification that is in the experimental cell. Alternatively, a control cell may be a parent of the experimental cell that is removed by one or more generations. Alternatively still, a control cell may be a sibling of the experimental cell, which sibling does not comprise the particular modification that is present in the experimental cell. A control cell can optionally be characterized as a cell as it existed before being modified to be an experimental cell.

[0083] The terms "sequence identity", "identity" and the like as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid residues or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, "percentage of sequence identity", "percent identity" and the like refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. It would be understood that, when calculating sequence identity between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered "identical" with, U residues of the RNA sequence. For purposes of determining "percent complementarity" of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and/or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.

[0084] Percent identity can be readily determined by any known method, including but not limited to those described in: 1) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991), all of which are incorporated herein by reference.

[0085] Preferred methods for determining percent identity are designed to give the best match between the sequences tested. Methods of determining identity and similarity are codified in publicly available computer programs, for example. Sequence alignments and percent identity calculations can be performed using the MEGALIGN program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.), for example. Multiple alignment of sequences can be performed, for example, using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values can correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method can be KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters can be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. Additionally the Clustal W method of alignment can be used (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992); Thompson, J. D. et al, Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (protein/nucleic acid) can be: GAP PENALTY=10/15, GAP LENGTH PENALTY=0.2/6.66, Delay Divergen Seqs(%)=30/30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.

[0086] Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced. Alternatively, a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function/activity of the disclosed sequence, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence. Any polypeptide amino acid sequence disclosed herein not beginning with a methionine can typically further comprise at least a start-methionine at the N-terminus of the amino acid sequence.

[0087] All the amino acid residues at each amino acid position of the proteins disclosed herein are examples. Given that certain amino acids share similar structural and/or charge features with each other (i.e., conserved), the amino acid at each position of a protein herein can be as provided in the disclosed sequences or substituted with a conserved amino acid residue ("conservative amino acid substitution") as follows:

[0088] 1. The following small aliphatic, nonpolar or slightly polar residues can substitute for each other: Ala (A), Ser (S), Thr (T), Pro (P), Gly (G);

[0089] 2. The following polar, negatively charged residues and their amides can substitute for each other: Asp (D), Asn (N), Glu (E), Gln (Q);

[0090] 3. The following polar, positively charged residues can substitute for each other: His (H), Arg (R), Lys (K);

[0091] 4. The following aliphatic, nonpolar residues can substitute for each other: Ala (A), Leu (L), Ile (I), Val (V), Cys (C), Met (M); and

[0092] 5. The following large aromatic residues can substitute for each other: Phe (F), Tyr (Y), Trp (W).

[0093] The terms "corresponds with", "corresponds to", "aligns with" and the like can be used interchangeably herein. The relative position of a conserved amino acid motif (e.g., SEQ ID NO:67 or 66) in an arabinose isomerase herein can, for example, correspond with certain positions/residues (e.g., positions 237-269) that are associated with (define the location of) the conserved motif as it exists in a reference arabinose isomerase (e.g., SEQ ID NO:7). The position of the conserved motif of SEQ ID NO:67 or 66 in a particular arabinose isomerase can thus be determined with reference to positions 237-269 of SEQ ID NO:7, for example. In general, one can align the amino acid sequence of a query arabinose isomerase with SEQ ID NO:7 using an alignment algorithm and/or software described herein (e.g., BLASTP, ClustalW, ClustalV, Clustal Omega, EMBOSS) to determine if the conserved motif of SEQ ID NO:67 or 66, if present, is located at the noted position. In some embodiments, an alignment further indicates that SEQ ID NO:67 or 66 is at a position corresponding with positions 237-269 of SEQ ID NO:7, if the location of SEQ ID NO:67 or 66 (i) begins at any residue from positions 217-257, 227-247, 230-244, or 232-242, and (ii) ends at any residue from positions 249-289, 259-279, 262-276, or 264-274, of the amino acid sequence of the query arabinose isomerase.

[0094] The term "isolated" as used herein refers to a polynucleotide or polypeptide molecule that has been completely or partially purified from its native source. In some instances, the isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix. For example, an isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner. Such a cell or organism containing heterologous components does not occur in nature, and/or exhibits properties not believed to naturally occur.

[0095] The term "increased" as used herein can refer to a quantity or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms "increased", "elevated", "enhanced", "greater than", "improved" and the like are used interchangeably herein. These terms can be used to characterize the "over-expression" or "up-regulation" of a polynucleotide encoding a protein, for example.

[0096] The present disclosure relates to engineered yeast strains that have arabinose isomerase activity. A challenge for engineering yeast to utilize arabinose, which is a sugar that can be obtained from cellulosic biomass, is to produce sufficient arabinose isomerase activity in the yeast cell. Arabinose isomerase catalyzes the conversion of arabinose to ribulose, which is the first step in an arabinose utilization pathway. Applicants have found that expression of specific arabinose isomerase polypeptides provides arabinose isomerase activity in yeast cells, while expression of other arabinose isomerase polypeptides does not provide activity. A yeast cell expressing arabinose isomerase activity provides a host cell for expression of a complete arabinose utilization pathway, thereby engineering a yeast cell that can produce a target chemical, such as ethanol, butanol, or 1,3-propanediol, using arabinose derived from lignocellulosic biomass as a carbon source, for example.

Yeast Host Cells

[0097] Yeast cells of the present disclosure are those that comprise an arabinose isomerase (i.e., heterologous arabinose isomerase) that supports effective utilization of arabinose in an arabinose utilization pathway, and are capable of producing a target chemical. Preferred target chemicals are those of commercial value including, but not limited to, ethanol, butanol, or 1,3-propanediol.

[0098] Any yeast cells that either produce a target chemical, or can be engineered to produce a target chemical, may be used as host cells herein. Examples of such yeasts include, but are not limited to, yeasts of the genera Kluyveromyces, Candida, Pichia, Hansenula, Schizosaccharomyces (e.g., S. pombe), Kloeckera, Schwanniomyces, Yarrowia, and Saccharomyces (e.g., S. cerevisiae).

[0099] Yeast cells of the present disclosure comprising an effective arabinose isomerase may be engineered according to methods well known in the art. For example, yeast cells that have the native ability to produce ethanol from C6 sugars may be transformed with genes encoding C5 metabolic pathways including an arabinose isomerase disclosed herein. Such cells may be capable of either aerobic or anaerobic fermentation ethanol production.

[0100] In some embodiments, yeast cells may be engineered to express a pathway for synthesis of butanol or 1,3-propanediol. Engineering of pathways for butanol synthesis (including isobutanol, 1-butanol, and/or 2-butanol) have been disclosed, for example, in U.S. Pat. Nos. 8,206,970 and 7,851,188, and in U.S. Patent Appl. Publ. Nos. 2007/0292927, 2009/0155870 and 2008/0182308, all of which are incorporated herein by reference. Engineering of pathways for 1,3-propanediol have been disclosed in U.S. Pat. Nos. 6,514,733, 5,686,276, 7,005,291, 6,013,494 and 7,629,151, which are incorporated herein by reference.

[0101] For utilization of xylose as a carbon source, a yeast cell can be engineered for expression of a complete xylose utilization pathway. Engineering of yeast such as S. cerevisiae for production of ethanol from xylose is described in Matsushika et al. (Appl. Microbiol. Biotechnol. 84:37-53) and in Kuyper et al. (FEMS Yeast Res. 5:399-409), which are incorporated herein by reference. In certain embodiments, in addition to engineering a yeast cell to have xylose isomerase activity, the activities of other pathway enzymes are increased in the cell to provide the ability to grow on xylose. Typically the activity levels of five pentose pathway enzymes are increased: xylulokinase (XKS1), transaldolase (TAL1), transketolase 1 (TKL1), D-ribulose-5-phosphate 3-epimerase (RPE1), and ribose 5-phosphate ketol-isomerase (RKI1). Any method known to one skilled in the art for increasing expression of a gene may be used. For example, as described in the Examples (below), these activities may be increased by expressing a coding region for each protein using a highly active promoter. Chimeric genes for expression can be constructed and integrated into the yeast genome. Alternatively, heterologous coding regions for these enzymes may be expressed in the yeast cell to obtain increased enzyme activities. Other suitable methods for engineering yeast capable of metabolizing xylose have been disclosed in, for example, U.S. Pat. Nos. 7,622,284, 8,058,040, 8,129,171 and 7,943,366, International Patent Appl. Publ. Nos. WO2011153516A2, WO2011149353A1, WO2006115455A1 and WO2011079388A1, and U.S. Patent Appl. Publ. Nos. 2010/0112658, 2010/0028975, 2009/0061502, 2007/0155000 and 2006/0216804, all of which are incorporated herein by reference.

[0102] For utilization of arabinose as a carbon source, a yeast cell can be engineered to express a complete arabinose utilization pathway, as disclosed in U.S. Patent Appl. Publ. No. 2005/0142648 and U.S. Pat. No. 8,129,171, for example, which are incorporated herein by reference. To allow arabinose utilization, activities expressed in addition to activities of the xylose utilization pathway include: 1) L-arabinose isomerase (examples of which are presently disclosed) to convert L-arabinose to L-ribulose, 2) L-ribulokinase to convert L-ribulose to L-ribulose-5-phosphate, and 3) L-ribulose-5-phosphate-4-epimerase to convert L-ribulose-5-phosphate to D-xylulose. These enzyme activities can be expressed using coding regions of araA, araB, and araD genes, respectively. In certain aspects, the araB-encoded L-ribulokinase is from E. coli, and the araD-encoded L-ribulose-5-phosphate-4-epimerase is from E. coli. Any method known to one skilled in the art for expressing a foreign coding region may be used. For example, as described in the Examples (below), these activities can be expressed by introducing chimeric genes containing promoters active in yeast cells, heterologous codon-optimized coding regions for the enzymes, and termination sequences active in yeast cells.

Arabinose Isomerase

[0103] Obtaining an effective amount of arabinose isomerase activity in yeast cells has been problematic. A group of arabinose isomerase enzymes were found herein that provide effective arabinose isomerase activity in a yeast cell for producing ethanol from arabinose in fermentation. The yeast cell, in addition to expressing the arabinose isomerase enzyme described herein, was genetically engineered as described above to express a xylose utilization pathway and a partial arabinose utilization pathway that lacks arabinose isomerase. The present arabinose isomerase was then expressed to complete the arabinose utilization pathway. One or more additional arabinose isomerases may also be expressed, if desired.

[0104] Twenty candidate arabinose isomerase enzymes (SEQ ID NOs:1-20) were chosen from the cow rumen metagenome dataset (Hess et al., Science 331:463-467) and the human microbiome dataset (The Human Microbiome Jumpstart Reference Strains Consortium et al., Science 328:994-999) as described in the Examples (below). Each of these arabinose enzymes was expressed in yeast cells from a codon-optimized coding sequence as described in Example 6 herein, and tested for the ability to support ethanol production by yeast cells grown in medium containing arabinose as the only sugar. Eight of the arabinose isomerase candidates (SEQ ID NOs:7, 8, 10, 15, 17, 18, 19 and 20) were effective in supporting production of ethanol, as compared to the arabinose isomerase from B. subtilis. Five of these arabinose isomerase candidates supported greater ethanol production as compared to the arabinose isomerase from B. subtilis: SEQ ID NOs:7, 10, 17, 18 and 19. Enzyme activities in protein extracts from the expressing strains showed these to be higher than for the B. subtilis arabinose isomerase, though the activities did not directly correlate with the ethanol production level of the corresponding strain in all cases. In some aspects, a yeast cell expressing an arabinose isomerase as presently disclosed can produce at least about 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the amount of ethanol that is produced under suitable conditions by a yeast cell expressing a B. subtilis arabinose isomerase (e.g., SEQ ID NO:41).

[0105] Six of the effective arabinose isomerase candidates (SEQ ID NOs:7, 8, 10, 17, 18 and 19) were found to be separated from the other candidates as members of one clade in a phylogenetic tree prepared for the twenty candidate arabinose isomerase enzyme amino acid sequences and the arabinose isomerase sequence from Bacillus subtilis (BSaraA; SEQ ID NO:41) (see FIG. 1). These six sequences were all from the cow rumen metagenome dataset. The sequences of SEQ ID NOs:7 and 8 have 94% amino acid sequence identity to each other. The sequences of SEQ ID NOs:10 and 17 have 92% amino acid sequence identity to each other. The sequences of SEQ ID NOs:10 and 17 have 80% amino acid sequence identity to SEQ ID NO: 18. Sequence identities among the other sequences are lower. Though the amino acid sequence identities vary among the six sequences of this clade, further sequence analysis identified an amino acid sequence motif (SEQ ID NO:67) that distinguishes the six sequences of this clade from the other fourteen candidate arabinose isomerase enzyme amino acid sequences, and that of the B. subtilis arabinose isomerase (SEQ ID NO:41). The distinguishing motif occurs starting with amino acid #237 and ending with amino acid #269, with reference to positions in the amino acid sequence of SEQ ID NO:7 (see FIGS. 2A and B). Based on the motif, this clade is called herein the "237-269 Motif" clade. The corresponding amino acid positions in different arabinose isomerase sequences can readily be determined by performing a sequence alignment. For example, these positions are #241 through #273 in both SEQ ID NOs:10 and 18.

[0106] The 237-269 Motif (FIG. 2A) is represented as formula I: I[RK]YQA[RK]EEIA[IM].K[IM][LM].[RA][EN]G[AC].AF.NTF[QE]DL . . . M (SEQ ID NO:67), where the standard single letter abbreviations for amino acids are used; where "[ ]" (brackets) indicate a position where the amino acid can be any one of the bracketed amino acids; and where each "." (period mark) indicates a position that does not have distinguishing amino acids (i.e., the residue at such position may also be found at the corresponding position in a non-clade AI sequence) (e.g., can be any standard amino acid) in terms of physical properties.

[0107] Further information about the 237-269 Motif (FIG. 2B) is represented in formula II: I[RK]YQA[RK]EEIA[IM].K[IM][LM].[RA][EN]G[AC].AF.NTF[QE]DL . . . M (SEQ ID NO:67), where the standard single letter abbreviations for amino acids are used; where "[ ]" (brackets) indicate a position where the amino acid can be any one of the bracketed amino acids; where the letters in bold indicate positions that are conserved (for the specific amino acid or for the physiochemical properties of the amino acid in each particular bold position) among all twenty candidate sequences and are therefore not distinguishing for the motif; where each "." (period mark) indicates a position that does not have distinguishing amino acids (i.e., the residue at such position may also be found at the corresponding position in a non-clade AI sequence) (e.g., can be any standard amino acid) in terms of physical properties; and where each underline indicates an amino acid that is only found at the underlined position in the present sequences (e.g., SEQ ID NOs:7, 8, 10, 17, 18, 19, or any other AI comprising SEQ ID NO:67) (i.e., they are not found in the non-clade sequences).

[0108] The amino acid sequence of the 237-269 Motif can be given as the following sequence, where the positions with non-distinguishing amino acids ("." positions) are shown as bracketed multiple amino acids, any one of which may occur in these bracketed positions:

TABLE-US-00002 (SEQ ID NO: 66) I[RK]YQA[RK]EEIA[IM][EK]K[IM][LM][VTD][RA][EN]G[AC] [KNR]AF[SVT]NTF[EQ]DL[HIY]GM.

Thus, the 237-269 Motif as represented by SEQ ID NO:66 has an "Xaa" at each position having possible multiple amino acids, with the possible amino acids in these positions, as shown above in the bracket positions, designated in the present Sequence Listing.

[0109] There is variation in the amino acids of the motif among the six sequences of the 237-269 Motif clade (see FIG. 2B), however, the amino acid sequence of an arabinose isomerase can be readily identified by one skilled in the art as belonging to this clade based on overall matching with the motif. Thus, in certain embodiments, the present arabinose isomerase sequences for expression in yeast have the 237-269 Motif described above (e.g., SEQ ID NO:67 or SEQ ID NO:66) or a related sequence (e.g., a motif that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:67 or SEQ ID NO:66). In some embodiments, an arabinose isomerase can comprise a motif that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to positions 237-269 of SEQ ID NO:7. The present arabinose isomerase sequences are any that belong to the 237-269 Motif clade (i.e., any that comprise SEQ ID NO:67, SEQ ID NO:66, or a related sequence thereof). It is noted for clarity that SEQ ID NO:66 is a version of SEQ ID NO:67.

[0110] In some embodiments, the present arabinose isomerase sequences are identified by specific amino acids matching to distinguishing positions in the motif sequence. The present arabinose isomerase is identified in this manner by having at least seventeen, eighteen, or nineteen of, or all of, the following amino acids in the motif: I at position 237; R or K at position 238; Y at position 239; R or K at position 242; E at position 243; I at position 245; A at position 246; I or M at position 247; K at position 249; I or M at position 250; R or A at position 253; E or N at position 254; G at position 255; A or C at position 256; F at position 259; N at position 261; T at position 262; Q or E at position 264; and M at position 269.

[0111] In some embodiments, the present arabinose isomerase sequences are identified by specific amino acids that are different from the amino acids at the corresponding positions in the sequences which are not in the 237-269 Motif clade. These specific amino acids are: E at position 243; I or M at position 250; A or C at position 256; and N at position 261.

[0112] In certain embodiments, an arabinose isomerase comprises, or consists of, an amino acid sequence that is 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO:7, 8, 10, 17, 18, or 19. Such an arabinose isomerase can optionally be further characterized to comprise SEQ ID NO:67 or SEQ ID NO:66 (or a variant of either motif that is at least 90% or 95% identical thereto), either of which being located at amino acid positions corresponding to positions 237-269 of SEQ ID NO:7. In some embodiments, an arabinose isomerase can (i) comprise, or consist of, an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO:7, 8, 10, 17, 18, or 19, and (ii) comprise the respective motif shown in FIG. 2C.

[0113] In some embodiments, an arabinose isomerase can comprise a motif that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to positions 237-269 of SEQ ID NO:7. Such an arabinose isomerase can optionally further be characterized as comprising, or consisting of, an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:7, 8, 10, 17, 18, or 19.

[0114] In some embodiments, an arabinose isomerase comprises, or consists of, the amino acid sequence of (i) SEQ ID NO:10, 17, 18, or 19 (or any variant thereof as disclosed herein) or (ii) SEQ ID NO:10, 17, or 18 (or any variant thereof as disclosed herein). An arabinose isomerase, in some aspects of the present disclosure, does not comprise SEQ ID NO:15, 68, 69, 70, or 71.

[0115] The present amino acid sequences that provide arabinose isomerase activity in yeast cells are not native to yeast cells, thus their encoding polynucleotide sequences are heterologous to yeast cells. For expression, polynucleotide molecules encoding the present polypeptides may be designed using codon optimization for the desired yeast cell. For example, to express SEQ ID NO:7, 8,10, 17, 18, and/or 19, a codon-optimized coding region of SEQ ID NO:27, 28, 30, 37, 38, and/or 39 can be used, respectively. A polynucleotide can also be characterized as being heterologous in some aspects by virtue of comprising heterologously combined elements (e.g., a promoter that is heterologous to the sequence encoding the polypeptide).

[0116] Methods for gene expression in yeasts are known in the art (see for example Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, Calif.). Expression of genes in yeast typically requires a promoter, operably linked to the coding region of interest, and a transcriptional terminator. A number of yeast promoters can be used in constructing expression cassettes for genes encoding the desired proteins, including, but not limited to, constitutive promoters (e.g., FBA1, GPD1, PDC1, ADH1, GPM, TPI1, TDH3, PGK1, ILV5p) and inducible promoters (e.g., GAL1, GAL10, CUP1). Suitable transcription terminators include, but are not limited to, FBAt, GPDt, GPMt, ERG10t, GAL1t, CYC1t, ADH1t, TAL1t, TKL1t, ILV5t, and ADHt.

[0117] A polynucleotide sequence herein encoding an arabinose isomerase can be a vector (e.g., plasmid, cosmid) containing a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host, for example. Typically used plasmids in yeast are shuttle vectors pRS423, pRS424, pRS425, and pRS426 (American Type Culture Collection, Rockville, Md.), which contain an E. coli replication origin (e.g., pMB1), a yeast 2.mu. origin of replication, and a marker for nutritional selection. The selection markers for these four vectors are His3 (vector pRS423), Trp1 (vector pRS424), Leu2 (vector pRS425) and Ura3 (vector pRS426). Additional vectors that may be used include pHR81 (ATCC #87541) and pRS313 (ATCC #77142). Construction of expression vectors with chimeric genes encoding the desired proteins may be performed by either standard molecular cloning techniques in E. coli or by the gap repair recombination method in yeast, for example.

[0118] The present disclosure also provides a method for producing a yeast cell that has arabinose isomerase activity following the teachings above. In this method, a heterologous polynucleotide encoding an arabinose isomerase as presently disclosed is introduced into a yeast cell lacking arabinose isomerase. Any yeast cell as disclosed herein can be produced using this method, if desired. Some aspects are drawn to increasing the arabinose isomerase activity of a yeast cell that already comprises a heterologous arabinose isomerase, by introducing a polynucleotide encoding an arabinose isomerase as presently disclosed to the yeast cell.

[0119] In various embodiments, a heterologous polynucleotide encoding a polypeptide having arabinose isomerase activity can be introduced into the yeast cell before, after, or at the same time that other genes for expressing enzymes of an arabinose utilization pathway are introduced.

[0120] In certain embodiments, a heterologous polynucleotide herein can be introduced into a yeast cell that has already been modified to comprise a complete xylose utilization pathway. Introduction of arabinose isomerase activity and additional modifications for a xylose utilization pathway and a complete arabinose utilization pathway may be performed in any order, and/or with two or more introductions/modifications performed concurrently. Such yeast cells have the ability to grow in medium containing arabinose as the sole carbon source. More typically, these cells are grown in medium containing arabinose, as well as other sugars such as glucose and/or xylose. This latter growth scheme allows effective use of the sugars found in a hydrolysate medium prepared from cellulosic biomass by pretreatment and saccharification. Some embodiments therefore are drawn to a fermentation comprising at least a yeast cell as disclosed herein, arabinose, and optionally xylose and/or glucose. Any or all of the sugar components of such a fermentation can be provided from a lignocellulosic biomass hydrolysate, for example.

[0121] In certain embodiments, a heterologous polynucleotide herein can be introduced into a yeast cell that has a metabolic pathway that produces a target chemical. The pathway may be endogenous, or it may be engineered in the cell. Introduction of arabinose isomerase activity and a metabolic pathway producing a target chemical may be performed in any order, and/or with two or more genetic modifications performed concurrently. Examples of target chemicals include ethanol, butanol, and 1,3-propanediol. Yeast cells containing metabolic pathways for production of target chemicals are described above, for example.

Production of a Target Chemical Using Arabinose

[0122] The present yeast cells expressing an arabinose isomerase as part of an arabinose utilization pathway, and producing ethanol and/or another target chemical, can be grown in medium containing arabinose. Typically, such yeast cells also are able to utilize xylose and the medium contains additional sugars such as glucose and xylose. In certain embodiments, lignocellulosic biomass hydrolysate is used in fermentation medium, for example as disclosed in U.S. Pat. No. 7,932,063, which is incorporated herein by reference.

[0123] A variety of culture methodologies may be applied. For example, large-scale fermentation/production may use batch, fed-batch, or continuous culture methodologies. Other fermentation conditions such as pH, oxygenation, and temperature can be applied, accordingly.

[0124] In any embodiment disclosed herein, an arabinose isomerase can, instead of being one as disclosed herein to comprise a 237-269 Motif (e.g., SEQ ID NO:66 or 67), rather be one comprising, or consisting of, an amino acid sequence that is 100% identical to, or at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, SEQ ID NO:15 or 20. Such enzymes do not belong to the 237-269 Motif clade.

[0125] Non-limiting examples of compositions and methods disclosed herein include:

1. A recombinant yeast cell comprising an arabinose utilization pathway that comprises a polypeptide having arabinose isomerase activity, wherein the yeast cell comprises a heterologous polynucleotide encoding the polypeptide, wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:67, and wherein the position of the motif in the polypeptide corresponds with positions 237-269 of SEQ ID NO:7. 2. The recombinant yeast cell of embodiment 1, wherein the motif comprises at least seventeen amino acids selected from the group consisting of: (a) I at position 237; (b) R or K at position 238; (c) Y at position 239; (d) R or K at position 242; (e) E at position 243; (f) I at position 245; (g) A at position 246; (h) I or M at position 247; (i) K at position 249; (j) I or M at position 250; (k) R or A at position 253; (l) E or N at position 254; (m) G at position 255; (n) A or C at position 256; (o) F at position 259; (p) N at position 261; (q) T at position 262; (r) Q or E at position 264; and (s) M at position 269; wherein each position of (a)-(s) corresponds with the respective position in positions 237-269 of SEQ ID NO:7. 3. The recombinant yeast cell of embodiment 1 or 2, wherein the polypeptide comprises a motif that is SEQ ID NO:67. 4. The recombinant yeast cell of embodiment 1, 2, or 3, wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:66. 5. The recombinant yeast cell of embodiment 4, wherein the polypeptide comprises a motif that is SEQ ID NO:66. 6. The recombinant yeast cell of embodiment 1, 2, 3, 4, or 5, wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:7, 8, 10, 17, 18, or 19. 7. The recombinant yeast cell of embodiment 1, 2, 3, 4, 5, or 6, further comprising a metabolic pathway that produces a target compound, optionally wherein the target compound is ethanol, butanol, or 1,3-propanediol. 8. A method for producing a yeast cell having arabinose isomerase activity, the method comprising: (a) providing a yeast cell lacking arabinose isomerase; and (b) introducing a heterologous polynucleotide into the yeast cell, wherein the heterologous polynucleotide encodes a polypeptide having arabinose isomerase activity, and wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:67. 9. The method of embodiment 8, wherein: (i) the yeast cell of step (a) comprises one or more polynucleotides encoding enzymes, except an arabinose isomerase, of an arabinose utilization pathway, or (ii) step (b) further comprises introducing, into the yeast cell, one or more polynucleotides encoding enzymes of an arabinose utilization pathway, wherein this further introduction is at the same time of, or after, the introducing the heterologous polynucleotide encoding the polypeptide having arabinose isomerase activity. 10. A method of producing a target compound from arabinose comprising: (a) providing the recombinant yeast cell of any one of embodiments 1-7; (b) growing the yeast cell of (a) in medium comprising arabinose, wherein the target compound is produced; and c) optionally isolating the target compound of (b). 11. The method of embodiment 10, wherein the target compound is ethanol, butanol, or 1,3-propanediol. 12. A recombinant yeast cell comprising an arabinose utilization pathway that comprises a polypeptide having arabinose isomerase activity, wherein the yeast cell comprises a heterologous polynucleotide encoding the polypeptide, wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:15 or 20. 13. The recombinant yeast cell of embodiment 12, further comprising a metabolic pathway that produces a target compound, optionally wherein the target compound is ethanol, butanol, or 1,3-propanediol. 14. A method of producing a target compound from arabinose comprising: (a) providing the recombinant yeast cell of embodiment 12 or 13; (b) growing the yeast cell of (a) in medium comprising arabinose, wherein the target compound is produced; and c) optionally isolating the target compound of (b). 15. A method for producing a yeast cell having arabinose isomerase activity, the method comprising: (a) providing a yeast cell lacking arabinose isomerase; and (b) introducing a heterologous polynucleotide into the yeast cell, wherein the heterologous polynucleotide encodes a polypeptide (i) having arabinose isomerase activity and (ii) comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:15 or 20.

EXAMPLES

[0126] The present disclosure is further exemplified in the following Examples. It should be understood that these Examples, while indicating certain preferred aspects herein, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the disclosed embodiments, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the disclosed embodiments to various uses and conditions.

Example 1

Plasmid Constructs for Xylose Utilization Pathway

[0127] To make xylose-utilizing yeast strains, a plasmid for increasing expression of the pentose pathway in Saccharomyces cerevisiae that was described as P5 Integration Vector in GRE3 in U.S. Pat. No. 8,669,076 (Example 1 therein), which is incorporated herein by reference, was used. This plasmid was renamed herein as pSX01 (SEQ ID NO:43; FIG. 3A). It contains a 12719-bp P5 transgene fragment having five chimeric genes (XKS1, TKL1, RKI1, RPE1, and TAL1) and a URA3 marker, flanked by a pair of homologous recombination fragments (HRF), GRE3-I (572-bp) and GRE3-II (541-bp). GRE3-I and GRE3-II direct integration of the transgene fragment into the GRE3 locus on chromosome 8 of the S. cerevisiae genome, between positions 323809 and 324118. Integration truncates the GRE3 coding sequence between nucleotides 401 and 710, which removes a 308-bp sequence from it. On the transgene fragment, TKL1, RKI1, RPE1, and TAL1 encode four pentose phosphate pathway enzymes: transketolase (EC 2.2.1.1), ribose-5-phosphate ketokisornerase (EC 5.3.1.6), ribulose-phosphate 3-epimerase (EC 5.1.3.1), and transaldolase (EC 2.2.1.2); XKS1 encodes a xylose assimilation pathway enzyme xylulokinase (EC 2.7.1.17); URA3 functions as a selection marker for transformation of the URA3-deletion strains. A pair of Lox elements was located at 5' and 3' ends of the URA3 marker so that it could be removed after integration of the transgene, when a Cre recombinase is introduced to transformants.

[0128] Xylose isomerase (EC 5.3.1.5) is a key enzyme for the xylose assimilation pathway in many bacteria and a few fungi. It is a slow enzyme with poor kinetic properties. It was previously disclosed in U.S. Pat. Nos. 8,114,974 and 8,093,037, which are incorporated herein by reference, that a synthetic xylA gene (herein named VDxylA) was expressed and functioned well in S. cerevisiae. VDxylA has the amino acid sequence shown in SEQ ID NO:44. A 1323-bp VDxylA coding sequence was synthesized using codon optimization for expression in S. cerevisiae (SEQ ID NO:45). It was then linked with a 868-bp PDC1 promoter and 110-bp UAS(FBA1) enhancer at its 5' end and a 623-bp ILV5 terminator at its 3' end, forming a 2966-bp chimeric expression cassette designated as UAS(FBA1)::PDC1p::VDxylA::ILV5t (SEQ ID NO:46).

[0129] The VDxylA cassette was constructed into three different integration plasmids, which targeted for three integration loci specifically. These plasmids had a 2653-bp common backbone sequence, which contained an E. coli replication origin (ORI) and ampicillin-resistance marker (AP.sup.r) for plasmid propagation in E. coli, as well as KasI sites at both ends. The transgene sequences had a structure of HRF-UNDxylA Cassette/HRF-DD/URA3 Cassette/HRF-D. They were connected with the backbone through KasI sites. In this transgene structure, HRF-U and HRF-D were two homologous recombination fragments able to integrate the transgene into the S. cerevisiae chromosome between the sequences corresponding to these two HRFs. The URA3 cassette provided a selective marker for integration. HRF-DD was a third homologous recombination fragment corresponding to a chromosomal sequence further downstream of HRF-D. After integration, this fragment was able to interact with the chromosomal copy of HRF-DD to loop-out the URA3 cassette and the HRF-D, thus leaving the VDxylA cassette between HRF-U and HRF-DD on the chromosome without a selective marker.

[0130] Plasmid pSX208 (SEQ ID NO:47; FIG. 3B) has a VDxylA cassette in its transgene fragment. Its three HRFs are 546-bp STB5U-U, 487-bp STB5U-D, and 386-bp STB5U-DD, corresponding to the S. cerevisiae chromosome-VIII from coordinates 457706 to 458251, from coordinates 458334 to 458820, and from coordinates 458836 to 459221, respectively. Therefore, during integration, the transgene fragment inserts into chromosome-VIII between coordinates 458251 and 458334. After recycling the URA3 marker, the VDxylA cassette was located between coordinates 458251 and 458836, that is, in an intergenic region upstream of the STB5 locus (YHR178W).

[0131] Plasmid pSX209 (SEQ ID NO:48; FIG. 4A) has a VDxylA cassette in its transgene fragment. Its three HRFs are 465-bp AAP1U-U, 476-bp AAP1U-D, and 522-bp AAP1U-DD, corresponding to S. cerevisiae chromosome-VIII from coordinates 203581 to 203117, from coordinates 202845 to 202370, and from coordinates 202362 to 201841, respectively. Therefore, during integration, the transgene fragment inserts into chromosome-VIII between coordinates 203117 and 202845. After recycling the URA3 marker, the VDxylA cassette from pSX209 was located between coordinates 203117 and 202362, that is, in an intergenic region upstream of the AAP1 locus (YHR047C).

[0132] Plasmid pSX210 (SEQ ID NO:49; FIG. 4B) has a VDxylA cassette in its transgene fragment. Its three HRFs are 437-bp PTC7U-U, 453-bp PTC7U-D, and 465-bp PTC7U-DD, corresponding to S. cerevisiae chromosome-VIII from coordinates 249702 to 250138, from coordinates 250199 to 250651, and from coordinates 250670 to 251134, respectively. Therefore, during integration, the transgene fragment inserts into chromosome-VIII between coordinates 250138 and 250199. After recycling the URA3 marker, the VDxylA cassette from pSX210 was located between coordinates 250138 and 250670, that is, in an intergenic region upstream of the PTC7 locus (YHR076W). Table 2 is a summary of plasmids pSX208, pSX209, and pSX210.

TABLE-US-00003 TABLE 2 Summary of pSX208, pSX209 and pSX210 Constructs. pSX208 pSX209 pSX210 Size 8601 bp 8645 bp 8537 bp Backbone 6376-427 6420-427 6312-427 STB5U-U 428-973 428-892 428-864 VDxylA Cassette 980-3945 899-3864 871-3836 STB5U-DD 3960-4345 3879-4400 3851-4315 URA3 Cassette 4472-5826 4527-5881 4442-5796 STB5U-D 5889-6375 5944-6419 5859-6311

Example 2

Development of Xylose-Utilization Strains

[0133] To make xylose-utilizing recombinant strains, the S. cerevisiae strain PXI3 was used as the recipient of transgenes. PXI3, also called BP1548, is a CEN.PK-based haploid laboratory strain derived from prototrophic diploid strain CBS 8272 (Centraalbureau voor Schimmelcultures [CBS] Fungal Biodiversity Centre, Netherlands), with a genotype of MAT.alpha. ura3.DELTA. his3.DELTA.. The strain was described in detail in Patent Appl. Publ. No. 2014/0178954 (Example 5 therein), which is incorporated herein by reference. Both native URA3 coding sequences were deleted by an approach similar to that described in Patent Appl. Publ. No. 2014/0178954.

[0134] The 12719-bp P5 transgene fragment (see Example 1), which included the homologous recombination regions GRE3-I and GRE3-II, was isolated from plasmid pSX01 by KasI digestion and transformed into the PXI3 strain using the FROZEN-EZ Yeast Transformation II Kit from Zymo Research (Irvine, Calif.). Transformants were selected on plates with CM/Gluc/-Ura (Teknova), which is a synthetic dropout (SD) medium lacking uracil. The URA3 marker was removed by introducing a CRE recombinase vector, pJT254 (SEQ ID NO:50), into the transformants. This vector was derived from pRS413 and the cre coding region (nt 2562 to 3593) was under the control of the GAL1 promoter (nt 2119 to 2561). Strains that could no longer grow on SD (-uracil) medium were selected. Further passages on YPD medium (Teknova) were used to cure the pJT254 plasmid. The resulting CEN-PK-based strain was named PX112. It has a P5 transgene fragment integrated into GRE3 locus as described in Example 1, with a genotype of MAT.alpha. ura3.DELTA. his3.DELTA. gre3.DELTA.:P5.

[0135] To integrate the xylA gene into the PX112 strain, the transgene fragments with the flanked HRFs were amplified by PCR from pSX208 or isolated from pSX209 and pSX210 by KasI digestion. The fragments were sequentially transformed into the strain to achieve multiple insertions. For one round of integration, the xylA transgene fragment was transformed into the recipient strain using the FROZEN-EZ Yeast Transformation II Kit from Zymo Research. Transformants were selected on CM/Gluc/-Ura Plates. Accurate integration was confirmed by PCR. Integration was followed by a recycling procedure to remove the URA3 marker. For this purpose, the transformant was grown in YPD broth (Teknova) overnight and then on a CM-FOA plate (6.7 g/L Sigma yeast nitrogen base without amino acids, 0.77 g/L Clontech dropout mix without uracil, 20 g/L glucose, 40 g/L uracil, 1 g/L 5-fluoro-orotic acid, 20 g/L agar) for two days. The survivors were streaked on both a YPD plate and a CM/Gluc/-Ura plate. A marker-free transformant was identified when it grew on the YPD plate but not on the CM/Gluc/-Ura plate. Marker removal was confirmed by PCR. When the transgene fragments of pSX208, pSX209, and pSX210 were integrated sequentially into PX112, a CEN-PK based haploid strain was obtained, which has a genotype of MAT.alpha. ura3.DELTA.::loxP his3.DELTA. gre3.DELTA.:P5 AAP1U.DELTA.::UAS(FBA1)-PDC1p-VDxylA-ILV5t PTC7U.DELTA.::UAS(FBA1)-PDC1p-VDxylA-ILV5t STB5U.DELTA.::UAS(FBA1)-PDC1p-VDxylA-1 LV5t. The resulting strain was named PXI68.

[0136] PXI68 was subjected to adaptation to speed up xylose fermentation. Adaptation was carried out in 4 mL YPX4 broth (10 g/L yeast extract, 20 g/L peptone, 4 g/L xylose) with a starting cell density at an OD.sub.600 value of 0.5. The strain was grown under micro-aerobic conditions at 32.degree. C. and 200 rpm shaking until approximately 5 doublings. Then it was passed to fresh YPX4 and grown under the same conditions. Adaptation was completed after 10 passages (approximately 50 doublings). Individual adapted strains were isolated and examined for improved xylose utilization using a standard mini-fermentation assay. This assay is described in Example 4 below, using YPX4 (YP medium containing 40 g/L xylose) as fermentation broth. The adaptation resulted in the improved strain, PXI82.

Example 3

Plasmid Constructs for Arabinose Utilization Pathway

[0137] To assemble a bacterium-type arabinose assimilation pathway in a xylose-utilizing strain so that the resultant strain is able to utilize glucose, xylose, and arabinose, three enzymes are required. L-arabinose isomerase (EC 5.3.1.4) is a key enzyme for the arabinose assimilation pathway in many bacteria, encoded by the araA gene. Similar to xylose isomerase, it is a slow enzyme with poor kinetic properties. The B. subtilis araA (BSaraA) gene-encoded arabinose isomerase has the amino acid sequence shown in SEQ ID NO:41. To express this araA, a 1491-bp BSaraA coding region was synthesized using codon optimization for expression in S. cerevisiae (SEQ ID NO:42). It was then linked with the 678-bp ADP promoter (ADHp) at its 5' end and with the 252-bp CYC1 terminator at its 3' end, forming a 2429-bp chimeric expression cassette called ADHp::BSaraA::CYC1t (SEQ ID NO:51) or BSaraA cassette. L-ribulokinase (EC 2.7.1.16) is the second enzyme in the bacterial arabinose assimilation pathway, encoded by the araB gene. The E. coli araB (ECaraB) gene-encoded ribulokinase has the amino acid sequence shown in SEQ ID NO:52. To express this araB, a 1701-bp ECaraB coding region was synthesized using codon optimization for expression in S. cerevisiae (SEQ ID NO:53). It was then linked with the 700-bp ILV5 promoter (ILV5p) at its 5' end and with the 500-bp PHO13 terminator (PHO13-3'UTR) at its 3' end, forming a 2907-bp chimeric expression cassette called ILV5p::ECaraB::PHO13-3'UTR (SEQ ID NO:54) or ECaraB cassette. L-ribulose-5-phosphate 4-epimerase (EC 5.1.3.4) is the third enzyme in the bacterial arabinose assimilation pathway, encoded by the araD gene. The E. coli araD (ECaraD) gene-encoded L-ribulose-5-phosphate 4-epimerase has the amino acid sequence shown in SEQ ID NO:55. To express this araD, a 696-bp ECaraD coding region was synthesized using codon optimization for expression in S. cerevisiae (SEQ ID NO:56). It was then linked with the 679-bp GPD promoter (GPDp) at its 5' end and with the 316-bp ADH1 terminator (ADP1t) at its 3' end, forming a 1691-bp chimeric expression cassette called GPDp::ECaraD::ADH1t (SEQ ID NO:57) or ECaraD cassette.

[0138] The ECaraB cassette was constructed into an integration plasmid, which targeted integration in the PHO13 locus (YDL236W, encoding for an alkaline phosphatase specific for p-nitrophenyl phosphate) on chromosome-IV, resulting in pSA0-B (SEQ ID NO:58; FIG. 5A). Similar to the integration plasmid described earlier, the backbone of this plasmid contained an E. coli replication origin (pBR322 ori) and ampicillin-resistance marker (AP.sup.r) for plasmid propagation in E. coli, as well as AscI and NotI sites at the ends. In the integration plasmid, transgene sequences had a structure of PHO13-U2/ECaraB Cassette/PHO13-3'UTR/URA3 Cassette/PHO13-D2. It was connected with the backbone through NotI and AscI sites. In this transgene structure, 500-bp PHO13-U2 and 500-bp PHO13-D2 were two homologous recombination fragments, corresponding to the chromosome-IV sequence from coordinates 31794 to 32294 and from coordinates 32735 to 33234, respectively. These sequences direct integration of the transgenes into chromosome-IV between the sequences corresponding to these two HRFs, which interrupted the PHO13 locus and deleted the first 439 nucleotides in the coding region. Within the transgene structure, the URA3 cassette provided a selective marker for integration. The PHO13-3'UTR not only served as a terminator for ECaraB but also was the third homologous recombination fragment corresponding to a chromosomal sequence further downstream of PHO13-D2, from coordinates 33235 to 33734. After integration, this fragment was able to interact with the chromosomal copy of PHO13-3'UTR to loop-out the URA3 Cassette and PHO13-D2, thus leaving the ECaraB cassette between PHO13-U2 and PHO13-3'UTR on chromosome-IV without a selective marker. The marker recycling of URA3 further removed the rest of the PHO13 coding sequence. Therefore, the entire PHO13 coding sequence was completely deleted.

[0139] The BSaraA cassette and ECaraD cassette were constructed into a high copy number shuttle vector, resulting in pSA503 (SEQ ID NO:59, FIG. 5B) that could support a high level transient expression of these transgenes. The backbone of this plasmid contained an E. coli replication origin (pBR322 ori) and ampicillin-resistance marker (AP.sup.r) for plasmid propagation in E. coli. It also contained an S. cerevisiae 2 micron replication sequence, and LEU2 and URA3 selection markers for plasmid propagation in S. cerevisiae. The BSaraA cassette was located downstream of the 2 micron element, with a unique 5' BamHI site. The ECaraD cassette was located downstream of the BSaraA cassette but in opposite orientation, with a unique 5' SacII site. The two cassettes were separated by a unique NotI site.

Example 4

Development of Arabinose-Utilization Strains

[0140] To make arabinose-utilizing recombinant strains, PX182 (prepared in Example 2) was used as the recipient of transgenes. The transgene fragment of PHO13-U2/ECaraB Cassette/PHO13-3'UTR/URA3 Cassette/PHO13-D2 was isolated from plasmid pSA0-B by NotI and AscI digestion and transformed into the PX182 strain using the FROZEN-EZ Yeast Transformation II Kit from Zymo Research (Irvine, Calif.). Transformants were selected on CM/Gluc/-Ura plates. Accurate integration was confirmed by PCR. Integration was followed by a recycling procedure to remove the URA3 marker. For this purpose, transformants were grown in YPD broth overnight and then on CM-FOA plates (6.7 g/L Sigma yeast nitrogen base without amino acids, 0.77 g/L Clontech dropout mix without uracil, 20 g/L glucose, 40 g/L uracil, 1 g/L 5-fluoro-orotic acid, 20 g/L agar) for two days. The survivors were streaked on both YPD plates and CM/Gluc/-Ura plates. A marker-free transformant was identified when it grew on a YPD plate but not on a CM/Gluc/-Ura plate, and was named PX182-araB. Marker removal was confirmed by PCR. To introduce the BSaraA cassette and the ECaraD cassette into the strain, shuttle vector pSA503 was transformed into the PX182-araB strain using the FROZEN-EZ Yeast Transformation II Kit from Zymo Research. Transformants were selected on CM/Gluc/-Ura Plates and named PX182-araBAD5030.

[0141] The PX182-araBAD5030 strain was tested for arabinose assimilation and fermentation capacity in a mini-shaking bottle fermentation under micro-aerobic conditions. Fermentation broth was YPA4 (YP medium containing 40 g/L arabinose). To assemble the fermentation, the strain was grown in 3 mL YPD culture at 32.degree. C. with shaking at 200 rpm overnight. Fresh cells were added into 5-mL NALGENE PETG diagnostics vials containing 4 mL fermentation broth, with a starting OD.sub.600 at 0.5. The PETG vials were sealed by screw caps mounted with a WHEATON 20-mm SEPTA PTEF red rubber pad within it. Micro-aerobic conditions were achieved by inserting BD 26G needles through the caps. Fermentation was conducted at 32.degree. C. with shaking at 200 rpm. At specified time intervals, 0.5 mL of culture was drawn through the needle using a syringe. One fifth of the collected culture (0.1 mL) was used for measurement of OD.sub.600. The rest of the culture was spun in a micro-centrifuge at 14000 rpm for 2 min. The supernatant was carefully collected by pipette, placed into a 0.22-.mu.m COSTAR SPIN-X Centrifuge Tube Filter (Corning Inc., Corning, N.Y.), and then passed through the filter by micro-centrifuging at 6000 rpm for 2 min. The flow-through was loaded into an AGILENT 250-4 vial insert within a 2-mL crimp vial and sealed. Xylose, ethanol (EtOH) and other metabolites were analyzed by running flow-through samples through a BIORAD AMINEX HPX-A7H ion exclusion column with 0.01 N H.sub.2SO.sub.4 at a speed of 0.6 mL/min at 55.degree. C. on an AGILENT 1100 HPLC system. Assay results (FIG. 6) showed that during 72-hr of fermentation, arabinose concentration in the broth was reduced from 38.5 g/L to 10.1 g/L (73.7% arabinose had been consumed). The arabinose consumption resulted in cell growth from OD.sub.600 value of 0.5 to 7.9, and supported production of 12.4 g/L ethanol. It confirmed that an arabinose assimilation pathway was assembled successfully in the PX182-araBAD5030 strain. This pathway, as combined with the pentose phosphate pathway engineered into the strain previously, was able to ferment arabinose to ethanol.

Example 5

Identification of AraA Candidates from Cow Rumen and Human Microbiome Databases

[0142] Arabinose isomerase is slow enzyme and functions in the first step of the bacterial-type arabinose assimilation pathway. To identify new bacterial arabinose isomerase candidates for expression testing in yeast, we used amino acid sequences of the arabinose isomerases from seven bacteria species: Bacillus subtilis (BS; SEQ ID NO:41), Escherichia coli (EC; SEQ ID NO:60), Bacillus licheniformis (BL; SEQ ID NO:61), Clostridium acetobutylicum (CA; SEQ ID NO:62), Leuconostoc mesenteroides (LM; SEQ ID NO:63), Lactobacillus plantarum (LP; SEQ ID NO:64), and Pediococcus pentosaceus (PP; SEQ ID NO:65) as queries in BLAST searches against translated open reading frames of the databases generated from the cow rumen metagenome dataset (Hess et al., Science 331:463-467) and the human microbiome dataset (The Human Microbiome Jumpstart Reference Strains Consortium et al., Science 328: 994-999). The putative open reading frames in the BLAST search results were first filtered by removing those entries with greater than 70% identity to any of the seven query amino acid sequences, or those entries containing one or more ambiguous nucleotide "N" in the nucleotide sequences. As all seven query arabinose isomerases have an L-arabinose isomerase protein domain (Arabinose_Isome, PFAM identifier PF02610) followed by an L-arabinose isomerase C-terminal protein domain (Arabinose_Iso_C, PFAM identifier PF11762), we further removed any open reading frames in the BLAST search results which did not contain both aforementioned protein domains in the same order. From the remaining search results, nine putative arabinose isomerases from among the sequences with the closest identities to B. subtilis arabinose isomerase were chosen from the cow rumen metagenome dataset (CR), and eleven were chosen from the human microbiome dataset (HM). These twenty arabinose isomerase (AI) candidates are listed and their sequence identity comparisons with the seven query arabinose isomerases are given in Table 3.

TABLE-US-00004 TABLE 3 Al Candidates and Their Sequence Comparison with Known Als. Data SEQ Percent Identity to Seven Known Als Al Candidate set ID NO BS EC BL CA LM LP PP HMPREF9412_4417 HM 1 64.9 59.8 69.1 56.7 51.6 51.8 50.6 POTG_01507 HM 2 63.2 59.0 67.5 55.2 52.3 52.2 52.2 HMPREF9374_3716 HM 3 59.6 55.8 63.8 52.7 52.3 51.5 49.7 DORFOR_01282 HM 4 57.6 51.3 58.8 52.4 52.0 51.6 51.9 HMPREF0994_04908 HM 5 56.7 52.8 57.4 49.6 48.6 48.3 48.4 NODE_4061684 CR 6 55.6 49.7 56.4 51.6 48.9 48.6 47.6 NODE_3664377 CR 7 52.0 50.2 56.9 51.6 51.3 49.6 50.0 NODE_458803 CR 8 50.9 48.6 55.8 50.5 50.0 48.3 49.6 NODE_3921064 CR 9 50.9 46.9 52.0 48.2 46.8 45.5 45.5 NODE_3693095 CR 10 50.3 50.0 52.6 51.4 48.0 48.3 47.6 DORLON_00938 HM 11 56.4 51.7 58.4 52.6 51.8 51.6 51.3 HMPREF9469_04726 HM 21 56.3 50.6 58.2 50.0 50.2 48.2 49.3 HMPREF9467_00216 HM 13 55.7 49.8 57.0 50.0 49.3 48.2 48.4 RTO_26010 HM 14 55.3 51.3 57.6 50.2 49.6 50.3 49.3 BRYFOR_08166 HM 15 55.1 49.5 57.4 51.1 49.0 47.8 48.4 RUMOBE_03031 HM 16 54.7 51.6 57.6 50.8 50.8 48.9 49.3 NODE_3658038 CR 17 50.3 50.5 53.1 52.0 49.0 49.1 48.5 NODE_4175755 CR 18 48.3 49.7 50.8 50.6 48.6 49.7 48.6 NODE_2588280 CR 19 48.3 47.6 51.1 49.3 49.9 50.2 49.5 NODE_3735508 CR 20 45.2 44.7 46.7 48.8 42.4 43.1 43.1

Example 6

Plasmid Constructs and Functional Studies of the Arabinose Isomerase Candidates

[0143] Synthetic coding sequences for the twenty arabinose isomerase (AI) candidates identified above were designed and synthesized using codon optimization for expression in S. cerevisiae. They were named araA-1 to araA-20 (SEQ ID NOs:21-40, respectively), corresponding to arabinose isomerase candidates with amino acid sequences of SEQ ID NOs:1-20, respectively. To test these arabinose isomerases, each synthetic coding region was constructed into pSA503 between the last nucleotide of the ADHp fragment and the PacI site in front of the CYC1t fragment, which accurately replaced the BSaraA coding sequence. This resulted in twenty plasmid constructs (from pSA503-1 to pSA503-20) that had sequences identical to pSA503 except for different araA coding regions. All twenty plasmid constructs were transformed into the PXI82-araB strain using the FROZEN-EZ Yeast Transformation II Kit from Zymo Research. Transformants were selected on CM/Gluc/-Ura Plates and named PX182-araBAD50301 to PX182-araBAD50320, corresponding to plasmid constructs pSA503-1 to pSA503-20, respectively. As a control, pSA503 was also transformed into PXI82-araB and selected on a CM/Glud-Ura Plate. Its transformants were named as PXI82-araBAD50300. The constructs and transformants are summarized in Table 4.

TABLE-US-00005 TABLE 4 Summary of Twenty Synthetic AraA Nucleotide Sequences and Their Transformants. Synthetic SEQ Size of Transformant Plasmid araA ID NO araA Encoded Al PXI82- pSA503 BSaraA 42 1491 nt BSAI araBAD50300 PXI82- psSA503-1 araA-1 21 1488 nt HMPREF9412_4417 araBAD50301 PXI82- psSA503-2 araA-2 22 1488 nt POTG_01507 araBAD50302 PXI82- psSA503-3 araA-3 23 1488 nt HMPREF9374_3716 araBAD50303 PXI82- psSA503-4 araA-4 24 1500 nt DORFOR_01282 araBAD50304 PXI82- psSA503-5 araA-5 25 1497 nt HMPREF0994_04908 araBAD50305 PXI82- psSA503-6 araA-6 26 1497 nt NODE_4061684 araBAD50306 PXI82- psSA503-7 araA-7 27 1434 nt NODE_3664377 araBAD50307 PXI82- psSA503-8 araA-8 28 1434 nt NODE_458803 araBAD50308 PXI82- psSA503-9 araA-9 29 1497 nt NODE_3921064 araBAD50309 PXI82- psSA503- araA-10 30 1464 nt NODE_3693095 araBAD50310 10 PXI82- psSA503- araA-11 31 1500 nt DORLON_00938 araBAD50311 11 PXI82- psSA503- araA-12 32 1497 nt HMPREF9469_04726 araBAD50312 12 PXI82- psSA503- araA-13 33 1497 nt HMPREF9467_00216 araBAD50313 13 PXI82- psSA503- araA-14 34 1500 nt RTO_26010 araBAD50314 14 PXI82- psSA503- araA-15 35 1497 nt BRYFOR_08166 araBAD50315 15 PXI82- psSA503- araA-16 36 1497 nt RUMOBE_03031 araBAD50316 16 PXI82- psSA503- araA-17 37 1476 nt NODE_3658038 araBAD50317 17 PXI82- psSA503- araA-18 38 1467 nt NODE_4175755 araBAD50318 18 PXI82- psSA503- araA-19 39 1467 nt NODE_2588280 araBAD50319 19 PXI82- psSA503- araA-20 40 1413 nt NODE_3735508 araBAD50320 20

[0144] To carry out the functional study for these twenty arabinose isomerases, three transformants were picked as replicas from each of twenty transformations and a control transformation. They were tested for arabinose assimilation and fermentation capacity in the mini-shaking bottle fermentation under micro-aerobic conditions described in Example 4. The fermentation broth used was YPA4 (YP medium containing 40 g/L arabinose). Cultures were grown, and samples were taken and analyzed as described. Fermentation assays for transformants from PX182-araBAD50301 to PX182-araBAD50315 and control transformant PX182-araBAD50300 were carried out for 72 hrs. Fermentations for PX182-araBAD50316 PX182-araBAD50320 were carried out for only 48 hrs because fermentation went fast for those transformants. Therefore, the 48-hr fermentation assays using to PXI82-araBAD50300 were also set up as controls. The assay result for each transformant was an average of fermentation data from three replicas. Ethanol production relative to that of PXI82-araBAD50300 that expressed B. subtilis arabinose isomerase is summarized in Table 5.

TABLE-US-00006 TABLE 5 Functional Assays of Twenty AI Candidates SEQ ID NO Ethanol Production: % Synthetic of encoded of PXI82- Transformant araA protein araBAD50300 PXI82-araBAD50300 BSaraA 41 100.0% PXI82-araBAD50301 araA-1 1 0.0% PXI82-araBAD50302 araA-2 2 0.0% PXI82-araBAD50303 araA-3 3 0.0% PXI82-araBAD50304 araA-4 4 39.9% PXI82-araBAD50305 araA-5 5 65.5% PXI82-araBAD50306 araA-6 6 17.4% PXI82-araBAD50307 araA-7 7 110.9% PXI82-araBAD50308 araA-8 8 97.7% PXI82-araBAD50309 araA-9 9 68.8% PXI82-araBAD50310 araA-10 10 105.2% PXI82-araBAD50311 araA-11 11 39.6% PXI82-araBAD50312 araA-12 12 0.0% PXI82-araBAD50313 araA-13 13 43.8% PXI82-araBAD50314 araA-14 14 0.0% PXI82-araBAD50315 araA-15 15 94.6% PXI82-araBAD50316 araA-16 16 0.0% PXI82-araBAD50317 araA-17 17 109.9% PXI82-araBAD50318 araA-18 18 152.4% PXI82-araBAD50319 araA-19 19 139.0% PXI82-araBAD50320 araA-20 20 96.1%

[0145] The results showed that six arabinose isomerase candidates, which were constructed into pSA503-1, pSA503-2, pSA503-3, pSA503-12, pSA503-14, and pSA503-16, did not support production of ethanol by S. cerevisiae at all; seven arabinose isomerase candidates, which were constructed into pSA503-4, pSA503-5, pSA503-6, pSA503-9, pSA503-11 and pSA503-13, functioned in S. cerevisiae but were 31.2% to 82.6% less effective in supporting ethanol production than B. subtilis arabinose isomerase. The other eight arabinose isomerase candidates, which were constructed into pSA503-7, pSA503-8, pSA503-10, pSA503-15, pSA503-17, pSA503-18, pSA503-19 and pSA503-20, performed similarly to, or better than, B. subtilis arabinose isomerase for ethanol production. It is interesting to note that all of the candidates in this group originated from the cow rumen dataset except for araA-15, which was from the human microbiome dataset. The best candidate was a cow rumen arabinose isomerase encoded by araA-18. It supported ethanol production up to 152.4% of that supported by the B. subtilis arabinose isomerase.

Example 7

In Vitro Activity Assay of the Top Arabinose Isomerase Candidates

[0146] Example 6 showed that the top performers in fermentation to produce ethanol included PX182-araBAD50307, PX182-araBAD50308, PX182-araBAD50310, PX182-araBAD50315, PX182-araBAD50317, PX182-araBAD50318, PX182-araBAD50319 and PX182-araBAD50320. To determine whether the arabinose isomerases expressed in these transformants were indeed highly active, the transformants were grown in 25 mL CM/Gluc/-Ura broth (6.7 g/L Sigma yeast nitrogen base without amino acids, 0.77 g/L CLONTECH dropout mix without uracil, 2% glucose) overnight at 32.degree. C. with 200 rpm shaking. At the same time, PX182-araBAD50313 and PX182-araBAD50316 were also grown up as representatives of the groups of arabinose isomerases supporting ethanol production at less than BSara or not at all, respectively. PX182-araBAD50300 was grown as a positive control. To prepare total soluble protein extract, overnight-grown cells with an OD.sub.600 value of 100 were collected and washed in 10 mL protein extraction buffer (PEB) (10 mM triethanolamine, pH 8.0, 10 mM MgSO.sub.4, 1 mM DTT, 5% glycerol). Cells were resuspended in 1 mL ice-cold PEB with Roche COMPLETE MINI EDTA-Free proteinase inhibitors (product #: 11836170001) and transferred into a tube containing 0.5-mm soda lime glass beads (BioSpec Products, Inc.). Total protein was extracted by beating cells in the tube using a BIO101 FP120 FASTPREP at setting 6 for 30 sec. Beating was repeated six times. Between the beatings, the tube was cooled down on ice for 2 min. Finally, protein extract was obtained by centrifugation and the protein concentration was determined using Coomassie Protein Assay Reagent (Thermo Scientific).

[0147] Arabinose isomerase activity in each protein extract was measured by the cysteine-carbazol method (Dische and Borefreund, J. Biol. Chem. 192:583-587). First, a 100-.mu.L assay reaction was assembled to include 10 mM MgSO.sub.4, 10 mM triethanolamine (pH 8.0), 50 mM arabinose, and appropriate amount (2-10 .mu.L) protein extract. The reaction was incubated at 32.degree. C. for 10 min and then stopped by adding 3 mL ice cold 75% H.sub.2SO.sub.4. Ribulose produced in the assay was quantified in a color reaction by adding 100 .mu.L of 2.4% cysteine hydrochloride and 100 .mu.L of 0.12% carbazol ethanolic solution. After incubating at room temperature for 6 min, OD.sub.540 value was measured using a spectrophotometer. Ribulose concentration was determined by comparing the OD.sub.540 value with a standard curve of a ribulose color reaction. A unit of arabinose isomerase was defined as the amount of enzyme required to produce one micromole of ribulose in 10 minutes of incubation.

[0148] Arabinose isomerase activity of each protein extract was determined by the abovementioned cysteine-carbazol method. For one assay, three replicas were carried out and the result was an average of them. A blank assay without protein extract was set up and deducted from all assay results. Specific activity of a protein extract was calculated based on its activity unit and protein concentration, expressed as unit per milligram of protein per min. FIG. 7 shows arabinose isomerase activities in the protein extracts. The results indicated that (1) protein extract of PX182-araBAD50316 did not have detectable arabinose isomerase activity, which was consistent with its functional assay (Table 5); (2) protein extract of PX182-araBAD50313 presented an activity approximately 2.4-fold higher than that of PXI82-araBAD50300, even though in the functional assay the ethanol productivity of PX182-araBAD50313 was only 43.8% of PXI82-araBAD50300 (Table 5); (3) other protein extracts had activities up to about 20-fold higher than that of PXI82-araBAD50300, but the maximal ethanol productivity was about 152% of PXI82-araBAD50300 (Table 5). Thus ethanol production did not always directly correlate with the in vitro enzyme activity result, perhaps due to the different conditions of the intracellular environment. Good arabinose isomerase candidates should perform well not only in the in vitro activity assay but also in the functional assay. Therefore, both in vitro activity assay and fermentation functional assay confirmed that arabinose isomerase candidates expressed in PX182-araBAD50307, PX182-araBAD50308, PX182-araBAD50310, PX182-araBAD50315, PX182-araBAD50317, PX182-araBAD50318, PX182-araBAD50319, and PX182-araBAD50320 were the enzymes that performed as well or better than B. subtilis arabinose isomerase in S. cerevisiae.

Sequence CWU 1

1

711495PRTunknownsequence from Human Microbiome dataset 1Met Asn Leu Lys Pro His Thr Phe Trp Phe Val Thr Gly Ser Gln His1 5 10 15Leu Tyr Gly Pro Glu Thr Leu Glu Gln Val Ala Glu His Ser Arg Ile 20 25 30Val Ala Thr Glu Phe Asp Lys Asp Pro Val Phe Thr Tyr Pro Ile Val 35 40 45Phe Lys Pro Ile Val Thr Thr Pro Asp Glu Ile Tyr Lys Leu Ile Leu 50 55 60Glu Ala Asn Asn Asp Glu Ser Cys Ala Gly Ile Met Thr Trp Met His65 70 75 80Thr Phe Ser Pro Ala Lys Met Trp Ile Ala Gly Leu Ser Gln Leu Gln 85 90 95Lys Pro Leu Leu His Phe His Thr Gln Phe Asn Arg Asp Ile Pro Trp 100 105 110Glu Thr Ile Asp Met Asp Phe Met Asn Leu Asn Gln Ser Ala His Gly 115 120 125Asp Arg Glu Tyr Gly His Ile Gly Ala Arg Leu Gly Ile Ala Arg Lys 130 135 140Val Val Val Gly His Trp Glu Asp Gly Glu Val Arg Gly Ser Ile Ala145 150 155 160Gly Trp Met Arg Thr Ala Ala Ala Tyr Ala Glu Ser Arg Arg Leu Lys 165 170 175Val Ala Arg Phe Gly Asp Asn Met Arg Gln Val Ala Val Thr Glu Gly 180 185 190Asp Lys Val Glu Ala Gln Ile Lys Leu Gly Trp Ser Val Asn Gly Tyr 195 200 205Gly Ile Gly Asp Leu Val Gln Ser Met Asn Glu Val Gly Asp Glu Glu 210 215 220Val Lys Ala Leu Leu Asn Glu Tyr Ala Glu Ser Tyr Ser Ile Thr Lys225 230 235 240Glu Gly Leu Ser Asp Gly Pro Val Arg Asp Ser Ile Ala Tyr Gln Ala 245 250 255Arg Ile Glu Ile Ala Leu Arg Arg Phe Leu Glu Glu Gly Gly Phe Gly 260 265 270Ala Phe Thr Thr Thr Phe Glu Asp Leu His Gly Met Lys Gln Leu Pro 275 280 285Gly Leu Ala Val Gln Arg Leu Met Glu Ser Gly Tyr Gly Phe Gly Gly 290 295 300Glu Gly Asp Trp Lys Thr Ala Ala Leu Thr Arg Val Leu Lys Val Leu305 310 315 320Ala Asp Asn Lys Ser Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Phe 325 330 335Glu Pro Gly Asn His Met Ile Leu Gly Ser His Met Leu Glu Val Cys 340 345 350Pro Thr Ile Ala Leu Asp Lys Pro Thr Leu Glu Val His Pro Leu Gly 355 360 365Ile Gly Gly Lys Gly Asp Pro Ala Arg Leu Val Phe Asn Gly Gln Asp 370 375 380Gly Pro Ala Val Asn Ala Ser Leu Ile Asp Leu Gly His Arg Phe Arg385 390 395 400Leu Leu Val Asn Val Val Asp Gly Val Lys Val Glu Gln Pro Met Pro 405 410 415Lys Leu Pro Val Ala Arg Val Leu Trp Lys Pro Gln Pro Ser Leu Arg 420 425 430Glu Ser Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala His His Thr Val 435 440 445Leu Ser Tyr Ala Met Thr Ala Glu His Leu Ser Asp Trp Ala Glu Met 450 455 460Thr Gly Ile Glu Ala Val Val Ile Asp Lys Asp Thr Thr Ile Pro Arg465 470 475 480Phe Lys Asn Glu Leu Arg Trp Ser Glu Ala Ala Tyr Arg Leu Arg 485 490 4952495PRTunknownsequence from Human Microbiome dataset 2Met Lys Leu Lys Pro His Ser Phe Trp Phe Val Thr Gly Ser Gln His1 5 10 15Leu Tyr Gly Pro Glu Thr Leu Glu Glu Val Ala Gly His Ser Arg Ile 20 25 30Ile Ala Glu Gln Leu Asp Lys Asp Pro Ala Ile Gly Phe Pro Val Val 35 40 45Phe Lys Pro Ile Val Thr Thr Pro Asp Glu Ile Tyr Lys Leu Ile Leu 50 55 60Ala Ala Asn Gly Asp Glu Thr Cys Ala Gly Ile Ile Thr Trp Met His65 70 75 80Thr Phe Ser Pro Ala Lys Met Trp Ile Ala Gly Leu Ser Gln Leu Gln 85 90 95Lys Pro Leu Leu His Phe His Thr Gln Phe Asn Arg Asp Ile Pro Trp 100 105 110Glu Thr Ile Asp Met Asp Phe Met Asn Leu Asn Gln Ser Ala His Gly 115 120 125Asp Arg Glu Tyr Gly His Ile Gly Ala Arg Leu Gly Ile Asn Arg Lys 130 135 140Ile Val Val Gly His Trp Glu Asp Glu Glu Val Arg Ala Ser Leu Ala145 150 155 160Gly Trp Met Arg Thr Ala Val Ala Tyr Ala Glu Ser Arg Gln Leu Lys 165 170 175Val Ala Arg Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr Glu Gly 180 185 190Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Ser Val Asn Gly Tyr 195 200 205Gly Val Gly Asp Leu Val Gln Val Leu Asn Glu Val Thr Asp Ala Glu 210 215 220Ala Glu Ala Leu Leu Lys Glu Tyr Ala Glu Gln Tyr Thr Ile Thr Gln225 230 235 240Ala Gly Leu Ser Ser Gly Pro Ile Arg Asp Ser Ile Ala Tyr Gln Ala 245 250 255Lys Leu Glu Ile Ala Met Lys Arg Phe Leu Glu Gln Gly Gly Phe Gly 260 265 270Ala Phe Thr Thr Thr Phe Glu Asp Leu His Gly Leu Lys Gln Leu Pro 275 280 285Gly Leu Ala Val Gln Arg Leu Met Glu Ala Gly Tyr Gly Phe Gly Gly 290 295 300Glu Gly Asp Trp Lys Thr Ala Ala Leu Thr Arg Val Leu Lys Val Leu305 310 315 320Ala Asn Asn Lys Ser Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Phe 325 330 335Glu Pro Gly Asn His Met Ile Leu Gly Ala His Met Leu Glu Val Cys 340 345 350Pro Thr Ile Ala Ala Thr Lys Pro Thr Ile Glu Val His Pro Leu Gly 355 360 365Ile Gly Gly Lys Ala Asp Pro Ala Arg Met Val Phe Asp Gly Gln Ala 370 375 380Gly Pro Ala Val Asn Ala Ser Leu Val Asp Leu Gly His Arg Phe Arg385 390 395 400Leu Leu Val Asn Val Val Asp Gly Val Lys Val Glu Lys Pro Met Pro 405 410 415Lys Leu Pro Val Ala Arg Val Leu Trp Lys Pro Gln Pro Ser Leu Arg 420 425 430Glu Ser Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala His His Thr Val 435 440 445Leu Ser Tyr Ala Ile Thr Ala Glu Asn Leu Ser Asp Trp Ala Glu Met 450 455 460Val Gly Ile Glu Ala Val Ile Ile Asp Lys Asp Thr Ser Val Pro Arg465 470 475 480Phe Lys Asn Glu Leu Arg Trp Ser Asp Ala Ala Tyr Arg Leu Arg 485 490 4953495PRTunknownsequence from Human Microbiome dataset 3Met Gln Arg Thr Pro Tyr Glu Phe Trp Phe Val Thr Gly Ser Gln His1 5 10 15Leu Tyr Gly Ser Glu Ala Leu Ala Glu Val Ser Ser His Ser Arg Gln 20 25 30Ile Thr Gln Ala Phe Asn Glu Ala Asp Ser Ile Ser Phe Pro Ile Val 35 40 45Val Lys Pro Val Val Lys Thr Pro Glu Glu Ile Leu Gln Leu Cys Met 50 55 60Glu Ala Asn Ser Asp Glu Asn Cys Ala Gly Leu Ile Thr Trp Met His65 70 75 80Thr Phe Ser Pro Gly Lys Met Trp Ile Gly Gly Leu Ser Gln Leu His 85 90 95Lys Pro Leu Leu His Phe His Thr Gln Phe His Arg Glu Ile Pro Trp 100 105 110Asp Arg Ile Asp Met Asp Phe Met Asn Leu His Gln Ser Ala His Gly 115 120 125Asp Arg Glu Phe Gly Phe Ile Ala Thr Arg Leu Gly Ile Leu Arg Lys 130 135 140Glu Val Val Gly His Trp Arg Asp Glu Ala Val Gln Lys Arg Leu Ser145 150 155 160Asp Trp Met Arg Thr Ala Ile Ala Cys Leu Glu Gly Lys Lys Leu Lys 165 170 175Val Ala Arg Phe Gly Asp Asn Met Arg Arg Val Ala Val Thr Glu Gly 180 185 190Asp Lys Val Glu Ala Gln Ile Gln Phe Gly Trp Ser Ile Asn Gly Tyr 195 200 205Gly Val Gly Asp Leu Val Gln Arg Ile Thr Asp Ile Ser Asp Thr Ala 210 215 220Val His Gln Leu Phe Arg Glu Tyr Gln Glu Arg Tyr Asp Phe Pro Pro225 230 235 240Glu Ala Arg Glu Ala Gly Pro Ile Arg Asp Ser Ile Leu Glu Gln Ala 245 250 255Arg Ile Glu Leu Gly Leu Lys Leu Phe Leu Arg Glu Gly Gly Tyr Ser 260 265 270Ala Phe Thr Thr Thr Phe Glu Asp Leu His Gly Leu Lys Gln Leu Pro 275 280 285Gly Leu Ala Val Gln Arg Leu Met Ser Glu Gly Tyr Gly Phe Gly Ala 290 295 300Glu Gly Asp Trp Arg Thr Ala Gly Leu Leu Arg Met Met Lys Ile Met305 310 315 320Ala Asp Asn Glu Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Leu 325 330 335Glu Pro Gly Asn Glu Met Ile Leu Gly Ala His Met Leu Glu Val Cys 340 345 350Pro Thr Ile Ala Ala Gln Arg Pro Gly Ile Arg Val His Pro Leu Ser 355 360 365Ile Gly Gly Lys Ala Asp Pro Ala Arg Leu Val Phe Asp Gly Arg Pro 370 375 380Gly Pro Ala Leu Asn Val Ser Leu Ile Asp Leu Gly Asn Arg Phe Arg385 390 395 400Leu Leu Ile Asn Lys Val Asp Ala Val His Pro Lys Ser Ala Met Pro 405 410 415His Leu Pro Val Ala Arg Val Leu Trp Lys Pro Arg Pro Ser Leu His 420 425 430Asp Ser Ala Glu Ala Trp Met Tyr Ala Gly Gly Ala His His Thr Val 435 440 445Phe Ser Tyr His Val Thr Thr Glu Gln Leu Leu Asp Trp Ala Glu Trp 450 455 460Val Asp Met Glu Ala Leu Val Ile Asp Glu Gln Thr Ser Leu Ser Ser465 470 475 480Phe Arg Arg Gln Leu Lys Trp Asn Asp Ala Tyr Tyr Arg Ile Arg 485 490 4954499PRTunknownsequence from Human Microbiome dataset 4Met Leu Lys Thr Lys Asn Tyr Gln Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Ala His Val Ala Glu His Ala Lys 20 25 30Lys Ile Val Glu Ala Leu Asn Ala Ser Gly Asn Leu Pro Tyr Glu Val 35 40 45Val Trp Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Arg Thr Phe 50 55 60Asn Glu Ala Asn Thr Asp Glu Asn Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Phe 85 90 95Arg Lys Pro Leu Leu His Leu His Thr Gln Phe Asn Arg Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Phe Gly His Ile Phe Ser Arg Leu His Met Asn Arg 130 135 140Lys Val Val Val Gly Tyr Trp Ala Asp Glu Asp Val Gln Lys Gln Ile145 150 155 160Gly Ser Trp Met Arg Thr Ala Val Gly Val Val Glu Ser Ser His Ile 165 170 175Arg Val Met Arg Ile Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Glu Val Asp Ala 195 200 205Tyr Pro Val Asn Glu Ala Val Glu Ala Val Asn Ala Val Ser Gln Ala 210 215 220Asp Ile Asp Thr Leu Val Glu Glu Tyr Tyr Asp Lys Tyr Glu Ile Leu225 230 235 240Leu Glu Gly Arg Asp Glu Lys Glu Phe Arg Arg His Val Ala Val Gln 245 250 255Ala Gly Ile Glu Ile Gly Leu Glu Arg Phe Leu Glu Glu Asn Asn Tyr 260 265 270Gln Ala Ile Val Thr His Phe Gly Asp Leu Gly Gly Phe Lys Gln Leu 275 280 285Pro Gly Leu Ala Met Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Thr Gly Gly Met Lys Asp Ala Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Pro Gly Lys Glu Gly Ile Leu Glu Ala His 340 345 350Met Leu Glu Val Cys Pro Thr Ile Ala Asp Gly Lys Ile Ser Ile Lys 355 360 365Glu Gln Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ala Lys Glu Gly Pro Ala Ile Ala Ala Ser Leu Ile Asp Leu385 390 395 400Gly Asp Arg Phe Arg Leu Leu Ile Asn Glu Val Glu Cys Lys Lys Thr 405 410 415Glu Lys Pro Met Pro Lys Leu Pro Val Ala Thr Ala Phe Trp Thr Pro 420 425 430Lys Pro Asn Leu Lys Ile Gly Ala Gln Ser Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Ser Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Glu Ala Met Gly Ile Glu Ala Val Tyr Ile Asp Ala Asp465 470 475 480Thr Thr Ile Arg Gln Leu Lys Asn Glu Leu Arg Trp Asn Glu Leu Ala 485 490 495Tyr Arg Arg5498PRTunknownsequence from Human Microbiome dataset 5Met Lys Thr Gly Arg Asp Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Glu Glu Cys Leu Arg Lys Val Ala Glu His Ser Ala 20 25 30Lys Ile Val Glu Gly Leu Asn Ala Ser Gly Arg Leu Pro Phe Glu Val 35 40 45Val Leu Lys Pro Thr Leu Ile Asp Pro Ala Thr Ile Arg Arg Thr Leu 50 55 60Asn Glu Ala Asn Glu Asp Gly Glu Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Met Trp Ile Leu Gly Leu Lys Glu Tyr 85 90 95Arg Lys Pro Leu Cys His Leu His Thr Gln Phe Asn Glu Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Phe Gly His Met Val Ser Arg Met Gly Met Glu Arg 130 135 140Lys Ile Ile Val Gly His Trp Ala Asn Ala Glu Val Gln Glu Lys Ile145 150 155 160Gly Ser Trp Met Arg Thr Ala Ile Gly Ile Met Glu Ser Ser His Ile 165 170 175Arg Val Cys Arg Ile Gly Asp Asn Met Asn Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Glu Val Lys Phe Gly Trp Glu Ile Asp His 195 200 205Tyr Cys Val Asn Asp Ala Val Glu Tyr Val Asn Ala Val Ser Glu Gly 210 215 220Asp Val Asn Ala Leu Val Glu Glu Tyr Tyr Ser Lys Tyr Gln Ile Leu225 230 235 240Leu Glu Gly Arg Asp Pro Glu Glu Phe Arg Ala His Val Ala Ala Gln 245 250 255Ala Lys Ile Glu Ile Gly Leu Glu Lys Phe Leu Glu Asp Gly Asp Tyr 260 265 270His Ala Ile Val Thr His Phe Gly Met Leu Gly Gly Leu Gln Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Ala Ala Gly Val Pro Gly Ala Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Pro Gly Lys Glu Gly Ile Leu Gln Ala His 340 345 350Met Leu Glu Val Cys Pro Ser Ile Ala Glu Gly Pro Ile Ser Ile Lys 355 360 365Val Gln Pro Leu Ser Met Gly Asn Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ser Lys Thr Gly Pro Ala Val Ala Thr Ser Leu Val Asp Leu385 390 395 400Gly Asn Arg Phe Arg Leu Ile Ile Asn Ala Val Asp Cys Lys Lys Cys 405 410 415Glu Lys Glu Met Pro Lys Leu Pro Val Ala Thr Ala Phe Trp Thr Pro 420 425 430Gln Pro Asp Leu Ala Thr Gly Ala Gln Ala Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Thr Val

Asp Gln Met Val 450 455 460Asp Trp Ala Ala Ala Met Gly Ile Glu Ser Val Val Ile Asp Lys Asp465 470 475 480Thr Thr Ile Arg Asn Phe Lys Asn Glu Leu Arg Trp Asn Ser Ile Tyr 485 490 495Tyr Arg6498PRTunknownsequence from cow rumen metagenome dataset 6Met Ile Gln Thr Lys Ala Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Val Leu Arg His Val Ala Asp His Ser Lys 20 25 30Glu Ile Val Glu Glu Leu Asn Lys Ser Gly Ile Leu Pro Tyr Glu Val 35 40 45Val Trp Lys Pro Val Leu Ile Thr Asn Gln Leu Ile Arg Gln Thr Phe 50 55 60Asn Glu Ala Asn Ala Asp Asp Ser Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Phe 85 90 95Arg Lys Pro Leu Leu His Leu His Thr Gln Tyr Asn Glu Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ala Ala His 115 120 125Gly Asp Arg Glu Tyr Gly His Ile Val Ser Arg Met Gly Ile Glu Arg 130 135 140Lys Val Ile Ala Gly Tyr Trp Lys Asp Asn Glu Val Arg Ser Arg Ile145 150 155 160Ala Ser Trp Met Arg Thr Ala Val Gly Val Met Glu Ser Ser His Ile 165 170 175Arg Val Met Arg Val Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Glu Val Asp Thr 195 200 205Tyr Pro Val Asn Glu Ile Ala Asp Ser Val Ala Thr Val Ser Ala Ser 210 215 220Asp Val Asn Ala Leu Leu Asp Glu Tyr Tyr Asp Lys Tyr Glu Ile Ile225 230 235 240Leu Asp Gly Arg Asp Pro Asp Glu Phe Lys Lys His Val Ala Val Gln 245 250 255Ala Gln Ile Glu Leu Gly Phe Glu Arg Phe Leu Glu Glu Lys Asn Tyr 260 265 270Gln Ala Ile Val Thr His Phe Gly Asp Leu Gly Ala Leu Gly Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Val Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Thr Ser Gly Met Lys Asp Ala Lys Gly Thr Ser Met Leu Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Arg Gly Lys Glu Gly Ile Leu Glu Ala His 340 345 350Met Leu Glu Ile Cys Pro Thr Ile Ala Asp Gly Pro Ile Ser Ile Arg 355 360 365Val Lys Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ser Lys Glu Gly Lys Gly Val Ala Thr Ser Leu Ile Asp Leu385 390 395 400Gly Asn Arg Phe Arg Leu Ile Ile Asn Glu Val Glu Cys Lys Lys Thr 405 410 415Glu Lys Pro Met Pro Asn Leu Pro Val Ala Thr Ala Tyr Trp Thr Pro 420 425 430Tyr Pro Asp Leu Tyr Thr Gly Ala Glu Ala Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Thr Ser Gly Gln Met Ala 450 455 460Asp Trp Ala Glu Met Met Gly Ile Glu Ala Val Ile Ile Asp Lys Asn465 470 475 480Thr Thr Ile Pro Ala Phe Lys Lys Glu Leu Lys Leu Gly Asp Val Phe 485 490 495Tyr Arg7477PRTunknownsequence from cow rumen metagenome dataset 7Met Lys Phe Trp Phe Val Thr Gly Ser Gln Phe Leu Tyr Gly Glu Glu1 5 10 15Thr Leu Arg Gln Val Glu Glu Asp Ser Lys Lys Ile Val Asp Gly Leu 20 25 30Arg Leu Pro Phe Pro Val Glu Tyr Lys Leu Thr Val Lys Thr Glu Ser 35 40 45Glu Ile Glu Arg Ile Val Lys Glu Ala Asn Tyr Asp Asp Glu Cys Ala 50 55 60Gly Ile Ile Thr Phe Cys His Thr Phe Ser Pro Ser Lys Met Trp Ile65 70 75 80Asn Gly Leu Ala Leu Leu Gln Lys Pro Trp Leu His Phe His Thr Gln 85 90 95Phe Asn Glu Thr Ile Pro Asn Glu Ala Ile Asp Met Asp Tyr Met Asn 100 105 110Leu His Gln Ser Ala His Gly Asp Arg Glu His Gly Phe Ile Gly Ala 115 120 125Arg Leu Arg Val Pro Arg Ala Val Val Ala Gly Tyr Trp Lys Asp Pro 130 135 140Ala Val Gln Ala Lys Ile Gly Glu Trp Gln Arg Ala Ala Val Gly Val145 150 155 160Met Phe Ser Arg Ser Leu Lys Ile Val Arg Phe Gly Asp Asn Met Arg 165 170 175Glu Val Ala Val Thr Glu Gly Asp Lys Ile Glu Ala Gln Leu Arg Leu 180 185 190Gly Trp Gln Val Asn Thr Phe Ala Val Gly Asp Leu Val Glu Tyr Met 195 200 205Asp Ala Val Thr Asp Ala Glu Ile Asp Ala Leu Met Lys Glu Tyr Ala 210 215 220Glu Leu Tyr Glu Phe Ser Glu Ala Asp Thr Asp Thr Ile Arg Tyr Gln225 230 235 240Ala Arg Glu Glu Ile Ala Ile Glu Lys Ile Leu Val Arg Glu Gly Ala 245 250 255Lys Ala Phe Ser Asn Thr Phe Glu Asp Leu His Gly Met Lys Gln Leu 260 265 270Pro Gly Leu Ala Thr Gln His Leu Met His Lys Gly Tyr Gly Phe Gly 275 280 285Ala Glu Gly Asp Trp Lys Thr Ala Gly Met Thr Ala Ile Val Lys Ala 290 295 300Met Tyr Pro Asp Gly Asn Thr Ser Phe Met Glu Asp Tyr Thr Tyr Asp305 310 315 320Tyr Glu Arg Gln Leu Ile Leu Gly Ser His Met Leu Glu Val Cys Pro 325 330 335Ser Ile Ala Ala Asp Arg Pro Arg Ile Glu Val His Lys Leu Gly Ile 340 345 350Gly Gly Lys Asp Ala Pro Ala Arg Ile Val Phe Glu Gly Arg Ala Gly 355 360 365Ser Ala Lys Val Leu Ser Leu Ile Asp Ile Gly Gly Arg Phe Arg Leu 370 375 380Ile Gln Gln Asp Ile Glu Cys Glu Lys Pro Phe Gln Ser Met Pro Asn385 390 395 400Leu Pro Val Ala Arg Thr Met Trp Arg Pro Ala Pro Ser Phe Leu Glu 405 410 415Gly Leu Glu Cys Trp Ile Ile Ala Gly Gly Ala His His Thr Val Leu 420 425 430Ser Tyr Asp Ile Thr Asp Glu Thr Val Arg Asp Phe Ala Arg Ile Met 435 440 445Gly Ile Glu Leu Val Val Ile Asn Lys Asp Thr Thr Lys Glu Lys Leu 450 455 460Glu Arg Asp Ile Met Ile Gly Asp Val Ile Tyr Gly Arg465 470 4758477PRTunknownsequence from cow rumen metagenome dataset 8Met Lys Phe Trp Phe Ile Thr Gly Ser Gln Phe Leu Tyr Gly Glu Glu1 5 10 15Thr Ile Arg Gln Val Glu Glu Asp Ser Lys Lys Ile Val Asp Gly Leu 20 25 30Lys Leu Pro Phe Pro Val Glu Tyr Lys Leu Thr Val Lys Lys Glu Ser 35 40 45Glu Ile Glu Arg Ile Val Lys Glu Ala Asn Phe Asp Asp Glu Cys Ala 50 55 60Gly Ile Ile Thr Phe Cys His Thr Phe Ser Pro Ser Lys Met Trp Ile65 70 75 80Asn Gly Leu Ala Ile Leu Gln Lys Pro Trp Leu His Phe His Thr Gln 85 90 95Phe Asn Glu Thr Ile Pro Asn Glu Ala Ile Asp Met Ala Tyr Met Asn 100 105 110Leu His Gln Ser Ala His Gly Asp Arg Glu His Gly Phe Ile Gly Ala 115 120 125Arg Leu Arg Met Pro Arg Ala Val Val Ala Gly Tyr Trp Lys Asp Pro 130 135 140Glu Val Gln Ala Lys Ile Ala Glu Trp Gln Arg Ala Ala Val Gly Val145 150 155 160Met Phe Ser Lys Ser Leu Lys Ile Val Arg Phe Gly Asp Asn Met Arg 165 170 175Glu Val Ala Val Thr Glu Gly Asp Lys Ile Glu Ala Gln Leu Lys Leu 180 185 190Gly Trp Gln Val Asn Thr Phe Ala Val Gly Asp Leu Val Glu Tyr Met 195 200 205Asn Ala Val Thr Asp Ala Glu Ile Asp Val Leu Met Lys Glu Tyr Ala 210 215 220Glu Leu Tyr Asp Tyr Asp Lys Ala Asp Glu Glu Thr Ile Arg Tyr Gln225 230 235 240Ala Arg Glu Glu Ile Ala Ile Glu Lys Ile Leu Val Arg Glu Gly Ala 245 250 255Lys Ala Phe Ser Asn Thr Phe Glu Asp Leu His Gly Met Gln Gln Leu 260 265 270Pro Gly Leu Ala Thr Gln His Leu Met His Lys Gly Tyr Gly Phe Gly 275 280 285Ala Glu Gly Asp Trp Lys Thr Ala Gly Met Thr Ala Ile Val Lys Ala 290 295 300Met Tyr Pro Asp Gly Asn Thr Ser Phe Met Glu Asp Tyr Thr Tyr Asp305 310 315 320Tyr Glu Arg Lys Leu Ile Leu Gly Ser His Met Leu Glu Val Cys Pro 325 330 335Ser Ile Ala Ala Asp Arg Pro Arg Ile Glu Val His Pro Leu Gly Ile 340 345 350Gly Gly Lys Glu Pro Pro Ala Arg Ile Val Phe Glu Gly Lys Ala Gly 355 360 365Ser Ala Lys Val Leu Ser Leu Ile Asp Ile Gly Gly Arg Leu Arg Leu 370 375 380Ile Gln Gln Asp Ile Glu Cys Glu Lys Pro Phe Gln Ser Met Pro Asn385 390 395 400Leu Pro Val Ala Arg Thr Met Trp Arg Pro Ala Pro Ser Phe Leu Glu 405 410 415Gly Leu Glu Cys Trp Ile Ile Ala Gly Gly Ala His His Thr Val Leu 420 425 430Ser Tyr Asp Ile Ser Asp Glu Thr Val Arg Asp Phe Ala Arg Ile Met 435 440 445Gly Ile Glu Leu Val Val Ile Asn Lys Asp Thr Thr Lys Glu Lys Leu 450 455 460Glu Arg Asp Ile Met Ile Gly Asp Met Ile Tyr Gly Arg465 470 4759498PRTunknownsequence from cow rumen metagenome dataset 9Met Ser Glu Met Lys Lys Tyr Gln Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Ala His Val Ala Ala His Ser Lys 20 25 30Glu Met Val Glu Gly Leu Asn Lys Ser Gly Val Leu Pro Phe Glu Ile 35 40 45Val Trp Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Lys Thr Phe 50 55 60Asn Glu Ala Asn Asn Asp Pro Asn Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Phe 85 90 95Arg Lys Pro Leu Leu His Leu His Thr Gln Tyr Asn Glu Glu Ile Pro 100 105 110Tyr Ala Thr Met Asp Met Asp Phe Met Asn Glu Asn Gln Ala Ala His 115 120 125Gly Asp Arg Glu Tyr Ala His Ile Leu Ser Arg Met Arg Ile Glu Arg 130 135 140Lys Val Val Val Gly Phe Trp Lys Asp Ser Glu Val Gln Lys Lys Ile145 150 155 160Ala Ser Trp Met Arg Thr Ala Ile Gly Ile Met Glu Ser Ser His Ile 165 170 175Arg Val Cys Arg Val Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Leu Lys Phe Gly Trp Glu Ile Asp Ala 195 200 205Tyr Pro Val Asn Glu Ile Ala Glu Ala Val Ala Ala Val Ser Ala Ser 210 215 220Asp Thr Asn Ala Leu Val Asp Glu Tyr Tyr Ser Lys Tyr Asp Ile Cys225 230 235 240Leu Glu Gly Arg Asp Pro Glu Glu Phe Lys Lys His Val Ala Val Gln 245 250 255Ala Gln Ile Glu Ile Gly Phe Glu Arg Phe Leu Lys Glu Lys Asn Tyr 260 265 270Gln Ala Ile Val Thr His Phe Gly Asp Leu Gly Ala Leu Lys Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Val Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Ser Ala Gly Met Lys Asp Ala Lys Gly Ser Ser Met Leu Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Lys Gly Lys Glu Gly Ile Ile Gln Ala His 340 345 350Met Leu Glu Ile Cys Pro Ser Ile Ser Asp Gly Pro Ile Gln Ile Lys 355 360 365Cys Gln Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Gln Ser Lys Thr Gly Ala Gly Ile Ala Thr Ser Leu Ile Asp Leu385 390 395 400Gly Asn Arg Phe Arg Leu Ile Ile Gln Asp Val Glu Cys Lys Lys Val 405 410 415Glu Lys Pro Leu Pro Lys Leu Pro Thr Ala Ile Asn Phe Trp Thr Pro 420 425 430Gln Pro Asp Phe Tyr Thr Gly Thr Glu Ala Trp Leu Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Ile Thr Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Ala Ala Met Gly Ile Glu Ala Val Phe Ile Asp Lys Asn465 470 475 480Thr Asn Ile Arg Asp Phe Lys Lys Asp Leu Met Leu Gly Glu Val Phe 485 490 495Tyr Arg10487PRTunknownsequence from cow rumen metagenome dataset 10Met Gln Arg Glu Phe Trp Phe Ile Val Gly Ser Gln Phe Leu Tyr Gly1 5 10 15Gln Asp Val Leu Asp Thr Val Asp Ala Arg Ala Arg Glu Met Ala Ala 20 25 30Glu Leu Ser Lys Val Leu Pro Tyr Pro Leu Val Tyr Lys Val Thr Ala 35 40 45Lys Thr Asn Lys Glu Ile Ala Asp Thr Val Lys Glu Ala Asn Tyr Arg 50 55 60Asp Glu Val Met Gly Ile Val Thr Trp Cys His Thr Phe Ser Pro Ser65 70 75 80Lys Met Trp Ile Asn Gly Leu Val Asn Leu Gln Lys Pro Tyr Cys His 85 90 95Leu Ala Thr Gln Tyr Asn Arg Glu Leu Pro Asn Glu Glu Ile Asp Ile 100 105 110Asp Phe Met Asn Leu Asn Gln Ala Ala His Gly Asp Arg Glu His Gly 115 120 125Phe Ile Ala Ala Arg Leu Arg Met Pro Arg Lys Val Ile Ala Gly Tyr 130 135 140Trp Gln Asp Glu Lys Val His Lys Arg Leu Ser Asp Trp Met Lys Ala145 150 155 160Ala Val Gly Val Asp Val Ser Lys His Met Lys Val Met Arg Phe Gly 165 170 175Asp Asn Met Arg Glu Val Ala Val Thr Glu Gly Asp Lys Val Glu Thr 180 185 190Gln Ile Lys Leu Gly Trp Gln Val Asn Thr Trp Ala Val Gly Asp Leu 195 200 205Val Lys Glu Met Asn Asn Val Thr Glu Ala Glu Ile Asp Ala Leu Phe 210 215 220Ala Glu Tyr Glu Ala Gln Tyr Asp Ile Ala Thr Asp Asn Leu Ala Ala225 230 235 240Ile Arg Tyr Gln Ala Lys Glu Glu Ile Ala Met Lys Lys Met Leu Asp 245 250 255Arg Glu Gly Cys Lys Ala Phe Ser Asn Thr Phe Gln Asp Leu Tyr Gly 260 265 270Met Glu Gln Leu Pro Gly Leu Ala Ser Gln His Leu Met Ala Gln Gly 275 280 285Tyr Gly Tyr Gly Gly Glu Gly Asp Trp Lys Val Ser Ala Met Thr Ala 290 295 300Ile Leu Lys Ala Met Gly Glu Asn Gly Asn Gly Ala Ser Ala Phe Met305 310 315 320Glu Asp Tyr Thr Tyr His Leu Val Glu Gly Gln Glu Tyr Ser Leu Gly 325 330 335Ala His Met Leu Glu Val Cys Pro Ser Leu Ala Ala Asp Lys Pro Arg 340 345 350Ile Glu Thr His His Leu Gly Ile Gly Met Asn Glu Lys Asp Pro Ala 355 360 365Arg Leu Val Phe Glu Gly Lys Ala Gly Lys Gly Ile Val Thr Ser Leu 370 375 380Ile Asp Met Gly Gly Arg Met Arg Leu Ile Val Gln Asp Ile Glu Ala385 390 395 400Val Lys Pro Ile Leu Pro Met Pro Asn Leu Pro Val Ala Arg Val Met 405 410 415Trp Arg Ala Met Pro Asp Leu Thr Thr Gly Val Glu Cys Trp Ile Thr 420 425 430Ala Gly Gly Ala His His Thr Val Leu Ser Phe Asp Val Thr Pro Ala 435 440

445Met Leu Arg Asp Trp Ala Arg Met Met Asp Ile Glu Phe Val Tyr Ile 450 455 460Thr Lys Asp Thr Thr Pro Glu Glu Leu Glu Glu Glu Leu Leu Ile Lys465 470 475 480Asp Leu Val Trp Lys Leu Lys 48511499PRTunknownsequence from Human Microbiome dataset 11Met Leu Lys Thr Lys Asn Tyr Gln Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Ala His Val Ala Glu His Ser Lys 20 25 30Ile Ile Val Asp Ala Leu Asn Lys Ser Gly Asn Leu Pro Tyr Glu Val 35 40 45Val Trp Lys Pro Thr Met Ile Thr Asn Glu Val Ile Arg Lys Thr Phe 50 55 60Asn Glu Ala Asn Thr Asp Glu Asn Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Tyr 85 90 95Arg Lys Pro Leu Leu His Leu His Thr Gln Phe Asn Arg Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ala Ala His 115 120 125Gly Asp Arg Glu Tyr Gly His Ile Phe Ser Arg Leu Asn Met Glu Arg 130 135 140Lys Val Val Ala Gly Tyr Trp Glu Asp Glu Asp Val Gln Lys Gln Ile145 150 155 160Gly Ser Trp Met Arg Thr Ala Val Gly Val Val Glu Ser Ser His Val 165 170 175Arg Val Met Arg Val Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Glu Val Asp Ala 195 200 205Tyr Pro Val Asn Glu Val Val Glu Ala Val Asn Ala Val Ser Gln Ala 210 215 220Asp Ile Asp Thr Leu Val Glu Glu Tyr Tyr Asp Lys Tyr Asp Ile Leu225 230 235 240Leu Glu Gly Arg Asp Glu Lys Glu Phe Arg Glu His Val Ala Val Gln 245 250 255Ala Gly Ile Glu Leu Gly Phe Glu Arg Phe Leu Asp Glu Asn Asn Tyr 260 265 270Gln Ala Val Val Thr His Phe Gly Asp Leu Gly Gly Leu Lys Gln Leu 275 280 285Pro Gly Leu Ala Met Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Val Met Lys Ile305 310 315 320Met Thr Gln Gly Met Lys Asp Ala Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Ser Gly Lys Glu Gly Val Leu Glu Ala His 340 345 350Met Leu Glu Val Cys Pro Thr Ile Ala Asp Gly Lys Ile Ser Ile Lys 355 360 365Glu Gln Pro Leu Ser Met Gly Asn Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ser Lys Thr Gly Pro Ala Ile Ala Thr Ser Leu Ile Asp Leu385 390 395 400Gly Asp Arg Phe Arg Leu Ile Ile Asn Asp Val Asp Cys Lys Lys Thr 405 410 415Glu Lys Pro Met Pro Lys Leu Pro Val Ala Thr Ala Phe Trp Thr Pro 420 425 430Gln Pro Asn Leu Lys Val Gly Thr Glu Ala Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Thr Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Ala Cys Met Gly Ile Glu Ala Val Tyr Ile Asp Lys Asp465 470 475 480Thr Thr Ile Arg Gln Phe Lys Asn Glu Leu Leu Trp Asn Ser Val Ala 485 490 495Tyr Arg Lys12498PRTunknownsequence from Human Microbiome dataset 12Met Thr Gly Val Lys Asn Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Glu Glu Cys Leu Ala His Val Ala Glu His Ser Arg 20 25 30Ile Ile Val Glu Ser Leu Asn Arg Ser Gly Ile Leu Pro Tyr Glu Val 35 40 45Val Trp Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Arg Thr Phe 50 55 60Asn Glu Ala Asn Ala Asp Glu Glu Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Phe 85 90 95Arg Lys Pro Leu Met His Phe His Thr Gln Phe Asn Arg Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Tyr Gly His Met Val Thr Arg Met Gly Ile Glu Arg 130 135 140Lys Val Ile Val Gly His Trp Ser Asp Glu Lys Val Val Gly Arg Ile145 150 155 160Ala Gly Trp Met Arg Thr Ala Val Gly Ile Met Glu Ser Ser His Val 165 170 175Arg Val Val Arg Phe Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Val Lys Phe Gly Trp Glu Val Asp Ala 195 200 205Tyr Pro Val Asn Glu Leu Cys Gln Tyr Val Lys Ala Val Pro Lys Gly 210 215 220Asp Ile Thr Ala Leu Val Asp Glu Tyr Tyr Ser Lys Tyr Thr Ile Leu225 230 235 240Leu Glu Gly Arg Asp Pro Glu Glu Phe Lys Arg His Val Ala Val Gln 245 250 255Ala Gln Ile Glu Ala Gly Leu Glu Arg Phe Leu Val Glu Lys Asp Tyr 260 265 270His Ala Ile Val Thr His Phe Gly Asp Leu Gly Glu Leu Gln Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Ala Gln Gly Val Lys Asn Ala Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Pro Gly Lys Glu Gly Ile Leu Glu Ala His 340 345 350Met Leu Glu Val Cys Pro Ser Ile Ala Asp Gly Glu Ile Ser Ile Lys 355 360 365Val Asn Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ser Lys Thr Gly His Gly Ile Ala Thr Ser Leu Val Asp Leu385 390 395 400Gly Thr Arg Phe Arg Leu Ile Ile Asn Asp Val Glu Cys Arg Lys Thr 405 410 415Glu Lys Ala Met Pro Lys Leu Pro Val Ala Thr Ala Phe Trp Thr Pro 420 425 430Glu Pro Ser Leu Ala Thr Gly Ala Glu Ala Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Thr Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Glu Ser Met Gly Ile Glu Val Val Tyr Ile Asp Lys Asp465 470 475 480Thr Thr Ile Arg Gly Leu Lys Asn Glu Met Arg Trp Asn Gly Ala Val 485 490 495Tyr Arg13498PRTunknownsequence from Human Microbiome dataset 13Met Ile Ala Val Lys Asn Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Ala His Val Ala Glu His Ser Gly 20 25 30Ile Ile Val Asp Ser Leu Asn Lys Ser Gly Ile Leu Pro Tyr Glu Val 35 40 45Val Leu Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Arg Thr Phe 50 55 60Asn Glu Ala Asn Ala Asp Glu Glu Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Tyr 85 90 95Arg Lys Pro Leu Met His Phe His Thr Gln Phe Asn Gln Glu Ile Pro 100 105 110Tyr Asp Ser Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Tyr Gly His Met Val Thr Arg Met Gly Ile Glu Arg 130 135 140Lys Val Ile Val Gly His Trp Arg Asp Glu Lys Val Val Gly Arg Ile145 150 155 160Ala Ala Trp Met Arg Thr Ala Val Gly Ile Met Glu Ser Ser His Val 165 170 175Arg Val Ala Arg Phe Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Met Lys Phe Gly Trp Glu Val Asp Ala 195 200 205Tyr Pro Val Asn Glu Leu Ala Glu Tyr Val Lys Ala Val Pro Lys Gly 210 215 220Asp Ile Thr Ala Leu Val Asp Glu Tyr Tyr Ser Lys Tyr Thr Ile Leu225 230 235 240Leu Glu Gly Arg Asp Pro Glu Glu Phe Lys Arg His Val Ala Val Gln 245 250 255Ala Gln Ile Glu Ala Gly Leu Glu Lys Phe Leu Leu Glu Lys Asp Tyr 260 265 270His Ala Ile Val Thr His Phe Gly Asp Leu Gly Glu Leu Gln Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Thr Gln Gly Met Lys Asp Ala Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Pro Gly Lys Glu Gly Ile Leu Glu Ala His 340 345 350Met Leu Glu Val Cys Pro Thr Ile Ala Asp Gly Glu Ile Ser Ile Lys 355 360 365Ala Cys Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ser Lys Thr Gly His Gly Ile Ala Ala Ser Leu Val Asp Leu385 390 395 400Gly Thr Arg Phe Arg Leu Ile Ile Asn Asp Val Glu Cys Lys Lys Thr 405 410 415Glu Lys Pro Met Pro Lys Leu Pro Val Ala Thr Ala Phe Trp Thr Pro 420 425 430Glu Pro Asn Leu Ala Thr Gly Ala Glu Ser Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Thr Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Asp Ala Met Gly Ile Glu Thr Val Tyr Ile Asp Lys Asp465 470 475 480Thr Thr Ile Arg Gly Leu Lys Asn Glu Leu Arg Trp Asn Ala Ala Ala 485 490 495Tyr Arg14499PRTunknownsequence from Human Microbiome dataset 14Met Leu Lys Lys Lys Glu Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Ala His Val Ala Glu His Ala Lys 20 25 30Ile Ile Val Glu Lys Leu Asn Glu Ser Gly Val Leu Pro Tyr Glu Val 35 40 45Val Trp Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Lys Thr Phe 50 55 60Asn Glu Ala Asn Ile Asp Asp Glu Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Phe 85 90 95Arg Lys Pro Leu Leu His Leu His Thr Gln Phe Asn Met Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ser Ala His 115 120 125Gly Gly Arg Glu Phe Gly His Ile Phe Thr Arg Leu Gly Ile Glu Arg 130 135 140Lys Val Val Val Gly His Trp Ser Asp Glu Lys Val Gln Glu Lys Ile145 150 155 160Ala Ser Trp Met Arg Thr Ala Val Gly Val Ile Glu Ser Ser His Val 165 170 175Arg Val Met Arg Val Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Glu Val Asp Ala 195 200 205Tyr Pro Val Asn Glu Ile Ala Glu Ser Val Asp Ala Val Ser Ala Ala 210 215 220Asp Val Asn Thr Leu Val Glu Glu Tyr Tyr Asp Lys Tyr Glu Ile Leu225 230 235 240Leu Glu Gly Arg Asp Pro Glu Glu Phe Arg Lys His Val Ala Val Gln 245 250 255Ala Gln Ile Glu Leu Gly Phe Glu Arg Phe Leu Glu Glu Lys Asn Tyr 260 265 270Gln Ala Ile Val Thr His Phe Gly Asp Leu Gly Val Leu Lys Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Gln Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Ile Met Lys Ile305 310 315 320Met Thr Glu Gly Met Lys Asp Ala Lys Gly Thr Ser Met Leu Glu Asp 325 330 335Tyr Thr Tyr Asn Phe Val Pro Gly Lys Glu Gly Ile Leu Gln Ala His 340 345 350Met Leu Glu Ile Cys Pro Ser Ile Ala Asp Gly Pro Ile Ser Ile Lys 355 360 365Val Asn Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Phe Thr Ser Lys Glu Gly Lys Gly Ile Ala Thr Ser Leu Ile Asp Leu385 390 395 400Gly Asp Arg Phe Arg Leu Ile Ile Asn Thr Val Asp Cys Lys Lys Asn 405 410 415Glu Lys Pro Met Pro Lys Leu Pro Val Ala Thr Asn Phe Trp Thr Pro 420 425 430Glu Pro Asp Leu Ala Thr Gly Ala Glu Ala Trp Ile Leu Cys Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Ile Thr Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Ala Met Met Gly Ile Glu Ala Val Tyr Ile Asp Lys Asp465 470 475 480Thr Thr Ile Arg Asn Leu Lys Asn Glu Leu Arg Trp Asn Glu Leu Ala 485 490 495Phe Arg Lys15498PRTunknownsequence from Human Microbiome dataset 15Met Lys Ala Ala Lys Asp Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Ala His Val Ala Glu His Ser Arg 20 25 30Ile Ile Val Asp Ala Leu Asn Lys Ser Gly Val Leu Pro Tyr Glu Ile 35 40 45Val Trp Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Lys Thr Phe 50 55 60Asn Glu Ala Asn Ala Asp Glu Asn Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Gln Glu Phe 85 90 95Arg Lys Pro Leu Leu His Phe His Thr Gln Phe Asn Arg Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ala Ala His 115 120 125Gly Asp Arg Glu Tyr Gly His Ile Val Ser Arg Met Gly Ile Glu Arg 130 135 140Lys Ile Ile Val Gly Tyr Trp Glu Asp Arg Asp Val Gln Glu Lys Ile145 150 155 160Ala Ser Trp Met Leu Thr Ala Ile Gly Ile Met Glu Ser Ser His Ile 165 170 175Arg Val Cys Arg Ile Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Glu Ile Asp Ala 195 200 205Tyr Pro Val Asn Glu Ile Ala Glu Tyr Val Ala Ala Val Pro Gln Gly 210 215 220Glu Ile Asn Ala Leu Val Glu Glu Tyr Tyr Ser Lys Tyr Asp Ile Ile225 230 235 240Leu Glu Gly Arg Asp Pro Gln Glu Phe Arg Glu His Val Ala Val Gln 245 250 255Ala Gly Ile Glu Ile Gly Phe Glu Lys Phe Leu Glu Glu Lys Asn Tyr 260 265 270Gln Ala Ile Val Thr His Phe Gly Asp Leu Gly Ser Leu Lys Gln Leu 275 280 285Pro Gly Leu Ala Ile Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Thr Ala Gly Val Lys Asn Pro Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Pro Gly Lys Glu Gly Val Leu Glu Ala His 340 345 350Met Leu Glu Val Cys Pro Ser Val Ala Asp Gly Pro Ile Gly Ile Lys 355 360 365Val Cys Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Val 370 375 380Tyr Thr Ser Lys Thr Gly Pro Ala Ile Ala Thr Ser Leu Ile Asp Leu385 390 395 400Gly Asn Arg Phe Arg Leu Ile Ile Asn Glu Val Glu Cys Lys Lys

Val 405 410 415Glu Lys Pro Met Pro Lys Leu Pro Val Ala Thr Ala Phe Trp Thr Pro 420 425 430Tyr Pro Asp Leu Lys Thr Gly Ala Glu Ala Trp Ile Leu Ala Gly Gly 435 440 445Ala His His Thr Ala Phe Ser Tyr Asp Leu Thr Ala Glu Gln Met Gly 450 455 460Asp Trp Ala Ala Ala Met Gly Ile Glu Ala Val Tyr Ile Asp Lys Asp465 470 475 480Thr Thr Ile Arg Asn Phe Lys Arg Asp Leu Gln Leu Gly Asn Ile Val 485 490 495Tyr Arg16498PRTunknownsequence from Human Microbiome dataset 16Met Val Thr Gly Arg Asn Tyr Lys Phe Trp Phe Cys Thr Gly Ser Gln1 5 10 15Asp Leu Tyr Gly Asp Glu Cys Leu Arg Lys Val Ala Glu His Ser Arg 20 25 30Ile Ile Val Glu Glu Leu Asn Lys Ser Gly Val Leu Pro Phe Glu Leu 35 40 45Val Trp Lys Pro Thr Leu Ile Thr Asn Glu Leu Ile Arg Lys Thr Phe 50 55 60Asn Glu Ala Asn Ala Asp Asp Glu Cys Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Ser Trp Ile Leu Gly Leu Lys Glu Tyr 85 90 95Arg Lys Pro Leu Cys His Leu His Thr Gln Phe Asn Gln Glu Ile Pro 100 105 110Tyr Asp Thr Ile Asp Met Asp Phe Met Asn Glu Asn Gln Ser Ala His 115 120 125Gly Gly Arg Glu Tyr Gly His Ile Val Thr Arg Met Gly Ile Glu Arg 130 135 140Lys Val Ile Val Gly His Trp Ala Asp Lys Lys Val Gln Glu Arg Leu145 150 155 160Ala Ser Trp Met Arg Thr Ala Val Gly Ile Met Glu Ser Ser His Ile 165 170 175Arg Val Cys Arg Val Ala Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Phe Gly Trp Glu Val Asp Ala 195 200 205Tyr Pro Val Asn Glu Val Cys Asp Tyr Val Lys Asp Val Ser Lys Gly 210 215 220Asp Ile Asp Val Leu Val Glu Glu Tyr Tyr Asn Lys Tyr Asp Ile Leu225 230 235 240Phe Glu Gly Arg Asp Pro Glu Glu Phe Lys Arg His Val Ala Val Gln 245 250 255Ala Ala Ile Glu Ile Gly Phe Glu Arg Phe Leu Glu Glu Lys Asn Tyr 260 265 270Gln Ala Val Val Thr His Phe Gly Asp Leu Gly Gly Leu Gln Gln Leu 275 280 285Pro Gly Leu Ala Met Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr Ala Ala Met Val Arg Leu Met Lys Ile305 310 315 320Met Thr Ala Gly Val Lys Asp Ala Lys Gly Thr Ser Phe Met Glu Asp 325 330 335Tyr Thr Tyr Asn Leu Val Pro Gly Lys Glu Gly Ile Leu Gln Ser His 340 345 350Met Leu Glu Val Cys Pro Thr Ile Ala Asp Gly Lys Ile Gly Ile Lys 355 360 365Val Cys Pro Leu Ser Met Gly Asp Arg Glu Asp Pro Ala Arg Leu Phe 370 375 380Thr Ser Lys Thr Gly Pro Ala Val Ala Thr Ser Leu Val Asp Leu Gly385 390 395 400Asp Arg Phe Arg Leu Ile Ile Asn Asp Val Asp Cys Lys Lys Val Glu 405 410 415Lys Pro Met Pro Lys Leu Pro Val Gly Ser Ala Phe Trp Thr Pro Gln 420 425 430Pro Asp Leu Ala Thr Gly Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala 435 440 445His His Thr Ala Phe Ser Tyr Asp Leu Thr Ala Glu Gln Met Gly Asp 450 455 460Trp Ala Ala Ala Met Gly Ile Glu Ala Val Tyr Ile Asp Lys Asp Thr465 470 475 480Thr Ile Arg Asn Phe Lys Asn Glu Leu Arg Trp Asn Glu Val Ala Phe 485 490 495Arg Lys17491PRTunknownsequence from cow rumen metagenome dataset 17Met Glu Asp Ile Met Lys Arg Glu Phe Trp Phe Ile Val Gly Ser Gln1 5 10 15Phe Leu Tyr Gly Gln Asp Val Leu Asp Thr Val Asp Ala Arg Ala Lys 20 25 30Glu Met Ala Ala Glu Leu Ser Lys Val Leu Pro Tyr Pro Leu Val Tyr 35 40 45Lys Val Thr Ala Lys Thr Asn Lys Glu Ile Thr Asp Val Ile Lys Glu 50 55 60Ala Asn Tyr Arg Asp Glu Cys Ala Gly Ile Val Thr Trp Cys His Thr65 70 75 80Phe Ser Pro Ser Lys Met Trp Ile Asn Gly Leu Ala Asn Leu Gln Lys 85 90 95Pro Tyr Cys His Leu Ala Thr Gln Tyr Asn Lys Glu Ile Pro Asn Asp 100 105 110Glu Ile Asp Met Asp Phe Met Asn Leu Asn Gln Ala Ala His Gly Asp 115 120 125Arg Glu His Gly Phe Ile Ala Ala Arg Leu Arg Leu Pro Arg Lys Val 130 135 140Ile Ala Gly Phe Trp Gln Asp Glu Lys Ile His Lys Arg Leu Ser Asp145 150 155 160Trp Met Arg Ala Ala Val Gly Val Ala Val Ser Lys Lys Met Lys Val 165 170 175Met Arg Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr Glu Gly Asp 180 185 190Lys Val Glu Val Gln Thr Lys Leu Gly Trp Gln Val Asn Thr Trp Ala 195 200 205Val Gly Asp Leu Val Lys Glu Met Gly Lys Val Thr Glu Ala Glu Ile 210 215 220Asp Ala Leu Val Ala Glu Tyr Glu Ala Asn Tyr Asp Ile Ala Thr Asp225 230 235 240Asn Thr Ala Ala Ile Arg Tyr Gln Ala Arg Glu Glu Ile Ala Met Lys 245 250 255Lys Met Leu Asp Arg Glu Gly Cys Arg Ala Phe Thr Asn Thr Phe Gln 260 265 270Asp Leu Tyr Gly Met Glu Gln Leu Pro Gly Leu Ala Ser Gln His Leu 275 280 285Met Ala Gln Gly Tyr Gly Tyr Gly Gly Glu Gly Asp Trp Lys Val Ser 290 295 300Ala Met Thr Ala Ile Leu Lys Ala Met Gly Glu Asn Gly Asn Gly Ala305 310 315 320Ser Gly Phe Met Glu Asp Tyr Thr Tyr His Leu Val Glu Gly Gln Glu 325 330 335Tyr Ser Leu Gly Ala His Met Leu Glu Val Cys Pro Ser Leu Ala Ala 340 345 350Asp Lys Pro Arg Ile Glu Thr His His Leu Gly Ile Gly Met Asn Glu 355 360 365Lys Asp Pro Ala Arg Leu Val Phe Glu Gly Lys Ala Gly Lys Gly Ile 370 375 380Val Val Ser Leu Ile Asp Met Gly Gly Arg Leu Arg Leu Ile Val Gln385 390 395 400Asp Ile Glu Ala Val Lys Pro Ile Leu Pro Met Pro Asn Leu Pro Val 405 410 415Ala Arg Val Met Trp Arg Ala Met Pro Asp Leu Thr Thr Gly Val Glu 420 425 430Cys Trp Ile Thr Ala Gly Gly Ala His His Thr Val Leu Ser Tyr Asp 435 440 445Val Thr Ala Glu Gln Met Arg Asp Trp Ala Arg Met Met Asp Ile Glu 450 455 460Phe Val His Ile Thr Lys Asp Thr Thr Pro Glu Lys Leu Glu Glu Glu465 470 475 480Leu Leu Val Lys Asp Leu Val Trp Lys Leu Lys 485 49018488PRTunknownsequence from cow rumen metagenome dataset 18Met Ser Lys Glu Phe Trp Phe Val Val Gly Ser Gln Asp Leu Tyr Gly1 5 10 15Glu Glu Val Leu Lys Ile Val Ala Glu Arg Ala Ala Glu Met Ala Ala 20 25 30Trp Leu Ser Glu Lys Leu Pro Tyr Pro Leu Ile Tyr Lys Val Thr Ala 35 40 45Met Ser Ser Asn Gln Ile Thr Ser Val Met Lys Glu Ala Asn Phe Asp 50 55 60Asp Asn Cys Leu Gly Val Val Thr Trp Cys His Thr Phe Ser Pro Ser65 70 75 80Lys Met Trp Leu Thr Gly Leu Asp Leu Leu Gln Lys Pro Trp Cys His 85 90 95Phe Ala Thr Gln Tyr Asn Leu Glu Ile Pro Asn Glu Glu Ile Asp Met 100 105 110Asp Phe Met Asn Leu Asn Gln Ala Ala His Gly Asp Arg Glu His Gly 115 120 125Phe Ile Gly Ala Arg Leu Arg Lys Ala Arg Lys Val Val Ala Gly Tyr 130 135 140Trp Lys Asp Glu Lys Val Ile Ala Arg Leu Ala Glu Phe Gln Lys Val145 150 155 160Ala Val Gly Val Asp Ala Ser Lys His Met Lys Val Met Arg Phe Gly 165 170 175Asp Asn Met Arg Asp Val Ala Val Thr Glu Gly Asp Lys Val Glu Val 180 185 190Gln Lys Lys Leu Gly Trp Glu Val Asn Thr Trp Ala Val Gly Asp Leu 195 200 205Val Lys Glu Met Asn Ala Val Thr Asp Glu Glu Val Glu Ala Leu Phe 210 215 220Asn Glu Tyr Lys Ala Ser Tyr Asp Ile Asn Thr Asp Asn Ile Tyr Ala225 230 235 240Ile Lys Tyr Gln Ala Arg Glu Glu Ile Ala Ile Lys Lys Met Met Asp 245 250 255Arg Asn Gly Cys Lys Ala Phe Ser Asn Thr Phe Gln Asp Leu Tyr Gly 260 265 270Met Glu Gln Leu Pro Gly Leu Ala Ser Gln His Leu Met Ser Leu Gly 275 280 285Tyr Gly Tyr Gly Gly Glu Gly Asp Trp Lys Val Ser Ala Met Thr Ala 290 295 300Ile Leu Lys Ala Met Gly Glu Asn Gly Asn Gly Ala Ser Ala Phe Met305 310 315 320Glu Asp Tyr Thr Tyr His Leu Val Lys Gly His Glu Tyr Ser Leu Gly 325 330 335Ala His Met Leu Glu Val Cys Pro Ser Leu Ala Ala Asp Lys Pro Arg 340 345 350Ile Glu Thr His His Leu Gly Ile Gly Met Asn Glu Lys Asp Pro Ala 355 360 365Arg Leu Val Phe Glu Gly Lys Glu Gly Arg Gly Ile Val Ala Ser Leu 370 375 380Ile Asp Met Gly Gly Arg Leu Arg Leu Ile Val Gln Asp Ile Glu Ala385 390 395 400Val Lys Pro Ile Met Pro Met Pro Asn Leu Pro Val Ala Arg Val Met 405 410 415Trp Arg Ala Leu Pro Asp Leu Thr Asp Gly Val Glu Cys Trp Ile Thr 420 425 430Ala Gly Gly Ala His His Thr Val Leu Ser Tyr Asp Val Thr Pro Glu 435 440 445Met Met Arg Asp Phe Ala Lys Phe Met Asp Ile Glu Phe Val His Ile 450 455 460Asp Lys Asp Thr Thr Val Glu Lys Leu Glu Asp Glu Leu Met Val Lys465 470 475 480Asp Leu Val Trp Lys Met Lys Gly 48519488PRTunknownsequence from cow rumen metagenome dataset 19Met Gly Asn Lys Lys Asn Phe Trp Phe Val Val Gly Ser Gln Phe Leu1 5 10 15Tyr Gly Asn Glu Val Leu Glu Thr Val Ala Ala Arg Ala Gln Glu Met 20 25 30Ala Glu Lys Met Ser Lys Ser Leu Pro Tyr Glu Leu Lys Phe Lys Gly 35 40 45Ile Val Lys Thr Trp Asp Glu Ala Thr Gln Tyr Ala Lys Glu Ala Asn 50 55 60Phe Asp Asp Asn Cys Cys Gly Val Ile Thr Trp Cys His Thr Phe Ser65 70 75 80Pro Ser Lys Met Trp Ile Glu Ala Phe Arg Leu Leu Gln Lys Pro Leu 85 90 95Leu His Phe Ala Thr Gln Tyr Asn Arg Tyr Ile Pro Asp Lys Glu Ile 100 105 110Asp Met Asp Phe Met Asn Leu Asn Gln Ala Ala His Gly Asp Arg Glu 115 120 125His Gly Phe Ile Ile Ala Arg Met Arg Leu Gln Gln Lys Ile Val Thr 130 135 140Gly Phe Trp Glu Asp Gln Pro Val Leu Asp Glu Ile Gly Thr Trp Met145 150 155 160Arg Ala Ala Val Ala Tyr Asp Phe Ser Arg Asn Leu Arg Val Met Arg 165 170 175Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr Glu Gly Asp Lys Val 180 185 190Glu Ala Gln Ile Lys Phe Gly Trp Gln Val Asn Thr Trp Pro Val Gly 195 200 205Lys Leu Val Glu Glu Ile Gly Lys Val Thr Glu Glu Glu Val Asp Glu 210 215 220Leu Leu Lys Val Tyr Thr Asp Thr Tyr Glu Leu Ala Thr Asp Asp Ile225 230 235 240Glu Thr Ile Arg Tyr Gln Ala Arg Glu Glu Ile Ala Met Lys Lys Met 245 250 255Met Thr Ala Glu Gly Ala Asn Ala Phe Val Asn Thr Phe Gln Asp Leu 260 265 270Ile Gly Met Lys Gln Leu Pro Gly Ile Ala Ser Gln His Leu Met Ala 275 280 285Gln Gly Tyr Gly Tyr Gly Ala Glu Gly Asp Trp Lys Leu Ser Ala Leu 290 295 300Val Ser Ile Val Lys Lys Met Thr Glu Gly Met Thr Gly Gly Thr Ser305 310 315 320Phe Met Glu Asp Tyr Thr Tyr His Leu Asp Pro Asn Ala Glu Tyr Ala 325 330 335Leu Gly Ala His Met Leu Glu Val Cys Pro Ser Ile Ala Ala Asp Lys 340 345 350Pro Arg Ile Glu Val His Pro Leu Gly Ile Gly Asp Arg Glu Asp Pro 355 360 365Ala Arg Leu Val Phe Glu Gly His Glu Gly Asp Ala Val Val Val Thr 370 375 380Leu Ile Asp Met Gly Glu Arg Phe Arg Met Leu Val Gln Asp Ile His385 390 395 400Cys Val Lys Pro Ile Tyr Glu Met Pro Asn Leu Pro Val Ala Arg Val 405 410 415Met Trp Glu Gly Lys Pro Ser Leu Asn Glu Gly Leu Lys Met Trp Leu 420 425 430Met Ala Gly Gly Ala His His Ser Val Leu Ser Tyr Asp Ala Thr Pro 435 440 445Glu Met Leu Lys Asp Leu Ala Arg Met Met Asp Ile Glu Phe Val His 450 455 460Ile Thr Ala Asp Ser Lys Pro Glu Glu Phe Glu Lys Asp Leu Phe Phe465 470 475 480Ala Asp Leu Ala Trp Lys Leu Lys 48520470PRTunknownsequence from cow rumen metagenome dataset 20Met Lys Lys Ile Tyr Phe Ile Thr Gly Ser Gln Asp Leu Tyr Gly Glu1 5 10 15Asp Val Leu Lys Thr Val Ala Lys Asp Ser Gln Glu Met Val Asn Phe 20 25 30Leu Asp Glu Gln Val Gly Glu Arg Ala Glu Ile Glu Phe Leu Gly Val 35 40 45Val Arg Asp Ser Glu Ile Cys Leu Asp Phe Ile Leu Lys Ala Asn Phe 50 55 60Asp Lys Glu Cys Ile Gly Ile Ile Thr Trp Met His Thr Phe Ser Pro65 70 75 80Ala Lys Met Trp Ile Arg Gly Leu Lys Val Leu His Lys Pro Met Leu 85 90 95His Leu His Thr Gln Tyr Asn Glu Lys Leu Pro Tyr Asp Ser Ile Asp 100 105 110Met Asp Phe Met Asn Leu Asn Gln Ala Ala His Gly Asp Arg Glu Phe 115 120 125Gly Phe Ile Ala Ala Arg Met Asn Ile Lys Gln His Val Leu Ser Gly 130 135 140Tyr Tyr Lys Asn Lys Asp Phe Ile Glu Gly Val Lys Gln Tyr Ile Asp145 150 155 160Val Cys Leu Ser Ile Asp Ala Ala Lys Tyr Leu Arg Val Ala Met Phe 165 170 175Gly Ser Asn Met Arg Asp Val Ala Val Thr Asp Gly Asp Arg Val Gln 180 185 190Ser Glu Ile Asp Phe Gly Trp Asn Val Asn Tyr Tyr Gly Ile Gly Asp 195 200 205Leu Val Asp Ile Ile Asn Lys Val Lys Asp Glu Glu Ile Asp Ala Gln 210 215 220Phe Glu Glu Tyr Lys Lys Arg Tyr Thr Ile Asn Thr Thr Asn Ile Glu225 230 235 240Ala Ile Lys Glu Gln Ala Lys Tyr Glu Val Ala Leu Lys Lys Phe Ile 245 250 255Lys Lys Glu Asn Val Gln Ala Phe Thr Asp Asn Phe Gln Asp Leu His 260 265 270Gly Leu Lys Gln Leu Pro Gly Leu Ala Val Gln Asp Leu Met Gln Glu 275 280 285Gly Ile Ser Phe Gly Pro Glu Gly Asp Tyr Lys Thr Pro Ala Leu Leu 290 295 300Ala Thr Leu Leu Pro Met Thr Lys Tyr Arg Lys Gly Ala Thr Gly Phe305 310 315 320Ile Glu Asp Tyr Thr Tyr Asp Leu Ile Glu Gly Lys Glu Ile Glu Leu 325 330 335Gly Ser His Met Leu Glu Val Pro Pro Ser Phe Ala Thr Ser Lys Pro 340 345 350Glu Ile Gln Val Arg Pro Leu Ser Ile Gly Asp Lys Ala Ala Pro Ala 355 360 365Arg Leu Val Phe Asp Ser Ile Glu Gly Glu Gly Leu Gln Ile Thr Met 370 375

380Val Asp Met Gly Thr His Phe Arg Ile Ile Ala Ala Lys Ile Gln Leu385 390 395 400Val Lys Gln Pro Ala Pro Met Pro Lys Leu Pro Val Ala Arg Ile Met 405 410 415Trp Lys His Ile Pro Asn Phe Lys Ile Ser Thr Glu Ala Trp Met Leu 420 425 430Tyr Gly Gly Gly His His Ser Val Ile Thr Thr Ala Leu Thr Ile Glu 435 440 445Asp Ile Lys Leu Phe Ala Lys Leu Thr Gly Thr Glu Leu Cys Val Ile 450 455 460Asp Glu Asn Thr Lys Ile465 470211488DNAunknownSequence from the human microbiome dataset 21atgaacttga agccacacac cttctggttc gtcactggtt cccaacactt gtacggtcca 60gaaactttgg aacaagtcgc tgaacactcc agaatcgtcg ctactgaatt tgacaaggac 120ccagttttca cctacccaat cgtcttcaag ccaatcgtta ccactccaga tgaaatctac 180aagttgatct tggaagctaa caacgacgaa tcctgtgctg gtatcatgac ttggatgcac 240accttctctc cagctaagat gtggatcgct ggtttgtccc aattgcaaaa gccattgttg 300cacttccaca ctcaattcaa cagagatatc ccttgggaaa ccatcgacat ggatttcatg 360aacttgaacc aatctgctca cggtgacaga gaatacggtc acatcggtgc tagattgggt 420atcgctagaa aggttgtcgt tggtcactgg gaagacggtg aagtcagagg ttctatcgct 480ggttggatga gaaccgctgc tgcttacgct gaatccagaa gattgaaggt cgctagattc 540ggtgacaaca tgagacaagt cgctgttacc gaaggtgaca aggttgaagc tcaaatcaag 600ttgggttggt ctgtcaacgg ttacggtatc ggtgacttgg ttcaatccat gaacgaagtc 660ggtgacgaag aagttaaggc tttgttgaac gaatacgctg aatcttactc catcactaag 720gaaggtttgt ctgacggtcc agtcagagat tccatcgctt accaagctag aatcgaaatc 780gctttgagaa gattcttgga agaaggtggt ttcggtgctt tcaccactac cttcgaagac 840ttgcacggta tgaagcaatt gccaggtttg gctgttcaaa gattgatgga atctggttac 900ggtttcggtg gtgaaggtga ctggaagact gctgctttga ccagagtctt gaaggttttg 960gctgacaaca agtctacttc cttcatggaa gattacacct accacttcga accaggtaac 1020cacatgatct tgggttctca catgttggaa gtctgtccaa ctatcgcttt ggacaagcca 1080accttggaag ttcacccatt gggtatcggt ggtaaaggtg acccagctag attggtcttc 1140aacggtcaag atggtccagc tgttaacgct tctttgatcg acttgggtca cagattcaga 1200ttgttggtca acgtcgttga cggtgtcaag gttgaacaac caatgccaaa gttgccagtc 1260gctagagttt tgtggaagcc acaaccatct ttgagagaat ctgctgaagc ctggatcttg 1320gctggtggtg ctcaccacac tgttttgtct tacgctatga ccgctgaaca cttgtccgac 1380tgggctgaaa tgaccggtat cgaagctgtc gttatcgaca aggatactac catcccaaga 1440ttcaagaacg aattgagatg gtctgaagct gcttacagat tgagatga 1488221488DNAunknownSequence from the human microbiome dataset 22atgaagttga agccacactc tttctggttc gttactggtt cccaacactt gtacggtcca 60gaaaccttgg aagaagtcgc tggtcactcc agaatcatcg ctgaacaatt ggacaaggac 120ccagctatcg gtttcccagt tgtcttcaag ccaatcgtta ccactccaga cgaaatctac 180aagttgatct tggctgctaa cggtgacgaa acttgtgctg gtatcatcac ttggatgcac 240accttctctc cagctaagat gtggatcgct ggtttgtccc aattgcaaaa gccattgttg 300cacttccaca ctcaattcaa cagagacatc ccttgggaaa ccatcgacat ggatttcatg 360aacttgaacc aatctgctca cggtgacaga gaatacggtc acatcggtgc tagattgggt 420atcaacagaa agatcgttgt cggtcactgg gaagacgaag aagtcagagc ttctttggct 480ggttggatga gaactgctgt tgcttacgct gaatccagac aattgaaggt cgctagattc 540ggtgacaaca tgagagaagt tgctgtcacc gaaggtgaca aggttgaagc tcaaatcaag 600ttcggttggt ctgttaacgg ttacggtgtc ggtgacttgg ttcaagtctt gaacgaagtc 660accgatgctg aagctgaagc cttgttgaag gaatacgctg aacaatacac tatcacccaa 720gctggtttgt cttccggtcc aatcagagac tccatcgctt accaagccaa gttggaaatc 780gctatgaaga gattcttgga acaaggtggt ttcggtgctt tcaccactac cttcgaagac 840ttgcacggtt tgaagcaatt gccaggtttg gctgttcaaa gattgatgga agctggttac 900ggtttcggtg gtgaaggtga ctggaagact gctgctttga ccagagtttt gaaggtcttg 960gctaacaaca agtctacttc cttcatggaa gactacacct accacttcga accaggtaac 1020cacatgatct tgggtgctca catgttggaa gtttgtccaa ctatcgctgc tactaagcca 1080accatcgaag tccacccatt gggtatcggt ggtaaagctg acccagctag aatggttttc 1140gatggtcaag ctggtccagc tgttaacgct tctttggttg acttgggtca cagattcaga 1200ttgttggtca acgttgtcga tggtgttaag gtcgaaaagc caatgccaaa gttgccagtt 1260gctagagtct tgtggaagcc acaaccatct ttgagagaat ctgctgaagc ctggatcttg 1320gctggtggtg ctcaccacac tgttttgtct tacgctatca ccgctgaaaa cttgtccgac 1380tgggctgaaa tggttggtat cgaagctgtc atcatcgaca aggatacctc tgtcccaaga 1440ttcaagaacg aattgagatg gtccgacgct gcttacagat tgagatga 1488231488DNAunknownSequence from the human microbiome dataset 23atgcaaagaa ctccatacga attctggttc gtcactggtt cccaacactt gtacggttcc 60gaagctttgg ctgaagtctc ctcccactcc agacaaatca ctcaagcctt caacgaagct 120gactctatct ccttcccaat cgttgtcaag ccagttgtca agaccccaga agaaatcttg 180caattgtgta tggaagctaa ctctgacgaa aactgtgctg gtttgatcac ttggatgcac 240accttctctc caggtaaaat gtggatcggt ggtttgtccc aattgcacaa gccattgttg 300cacttccaca ctcaattcca cagagaaatc ccttgggaca gaatcgacat ggatttcatg 360aacttgcacc aatctgctca cggtgacaga gaatttggtt tcatcgctac cagattgggt 420atcttgagaa aggaagttgt cggtcactgg agagacgaag ctgttcaaaa gagattgtct 480gattggatga gaactgctat cgcttgtttg gaaggtaaaa agttgaaggt tgctagattc 540ggtgacaaca tgagaagagt tgctgtcacc gaaggtgaca aggtcgaagc tcaaatccaa 600ttcggttggt ctatcaacgg ttacggtgtt ggtgacttgg tccaaagaat cactgacatc 660tccgataccg ctgttcacca attgttcaga gaataccaag aaagatacga cttcccacca 720gaagctagag aagctggtcc aatcagagat tctatcttgg aacaagctag aatcgaattg 780ggtttgaagt tgttcttgag agaaggtggt tactccgctt tcaccactac cttcgaagac 840ttgcacggtt tgaagcaatt gccaggtttg gctgtccaaa gattgatgtc tgaaggttac 900ggtttcggtg ctgaaggtga ctggagaact gctggtttgt tgagaatgat gaagatcatg 960gctgacaacg aaggtacttc cttcatggaa gattacacct accacttgga accaggtaac 1020gaaatgatct tgggtgctca catgttggaa gtttgtccaa ccatcgctgc tcaaagacca 1080ggtatcagag tccacccatt gtctatcggt ggtaaagctg acccagctag attggttttc 1140gatggtagac caggtccagc tttgaacgtc tctttgatcg acttgggtaa cagattcaga 1200ttgttgatca acaaggttga tgctgtccac ccaaagtccg ctatgccaca cttgccagtt 1260gctagagtct tgtggaagcc aagaccatct ttgcacgact ctgctgaagc ctggatgtac 1320gctggtggtg ctcaccacac tgttttctct taccacgtca ctaccgaaca attgttggac 1380tgggctgaat gggttgatat ggaagccttg gttatcgacg aacaaacctc cttgtcttcc 1440ttcagaagac aattgaagtg gaacgacgct tactacagaa tcagatga 1488241500DNAunknownSequence from the human microbiome dataset 24atgttgaaga ctaagaacta ccaattctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaatgtt tggctcacgt tgctgaacac gctaagaaga tcgttgaagc cttgaacgct 120tctggtaact tgccatacga agttgtctgg aagccaacct tgatcactaa cgaattgatc 180agaagaacct tcaacgaagc taacactgac gaaaactgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagagttcag aaagccattg 300ttgcacttgc acacccaatt caacagagaa atcccatacg acactatcga catggatttc 360atgaacgaaa accaatctgc tcacggtgac agagaatttg gtcacatctt ctccagattg 420cacatgaaca gaaaggttgt cgttggttac tgggctgacg aagatgttca aaagcaaatc 480ggttcttgga tgagaaccgc tgttggtgtc gttgaatctt cccacatcag agtcatgaga 540atcgctgaca acatgagaaa cgtcgctgtt actgaaggtg acaaggttga agctcaaatc 600aagttcggtt gggaagttga cgcttaccca gtcaacgaag ctgtcgaagc tgttaacgct 660gtctcccaag ctgacatcga taccttggtc gaagaatact acgacaagta cgaaatcttg 720ttggaaggta gagatgaaaa ggagttcaga agacacgtcg ctgttcaagc tggtatcgaa 780atcggtttgg aaagattctt ggaagaaaac aactaccaag ctatcgttac tcacttcggt 840gacttgggtg gtttcaagca attgccaggt ttggctatgc aaagattgat ggaaaagggt 900tacggtttcg gtgctgaagg tgactggaag accgctgcta tggtcagatt gatgaagatc 960atgactggtg gtatgaagga cgctaagggt acttctttca tggaagatta cacttacaac 1020ttggttccag gtaaagaagg tatcttggaa gctcacatgt tggaagtctg tccaaccatc 1080gctgacggta aaatctctat caaggaacaa ccattgtcta tgggtgacag agaagatcca 1140gctagattgg ttttcactgc taaggaaggt ccagctatcg ctgcttcttt gatcgacttg 1200ggtgacagat tcagattgtt gatcaacgaa gttgaatgta agaagaccga aaagccaatg 1260ccaaagttgc cagtcgctac cgctttctgg actccaaagc caaacttgaa gatcggtgct 1320caatcctgga tcttggctgg tggtgctcac cacactgctt tctcttacga cttgtccgct 1380gaacaaatgg gtgactgggc tgaagctatg ggtatcgaag ctgtctacat cgacgctgat 1440accactatca gacaattgaa gaacgaattg agatggaacg aattggctta cagaagatga 1500251497DNAunknownSequence from the human microbiome dataset 25atgaagactg gtagagatta caagttctgg ttctgtactg gttcccaaga tttgtacggt 60gaagaatgtt tgagaaaggt tgctgaacac tccgctaaga tcgtcgaagg tttgaacgct 120tctggtagat tgccattcga agttgtcttg aagccaacct tgatcgatcc agctaccatc 180agaagaactt tgaacgaagc taacgaagac ggtgaatgtg ctggtgttat cacctggatg 240cacactttct ccccagctaa gatgtggatc ttgggtttga aggaatacag aaagccattg 300tgtcacttgc acacccaatt caacgaagaa atcccatacg atactatcga catggatttc 360atgaacgaaa accaatccgc tcacggtgac agagaatttg gtcacatggt ttccagaatg 420ggtatggaaa gaaagatcat cgtcggtcac tgggctaacg ctgaagttca agaaaagatc 480ggttcttgga tgagaaccgc tatcggtatc atggaatctt cccacatcag agtctgtaga 540atcggtgaca acatgaacaa cgttgctgtc actgaaggtg acaaggtcga agctgaagtc 600aagttcggtt gggaaatcga tcactactgt gttaacgacg ctgttgaata cgtcaacgct 660gtttccgaag gtgacgtcaa cgctttggtt gaagaatact actctaagta ccaaatcttg 720ttggaaggta gagacccaga agagttcaga gctcacgtcg ctgctcaagc taagatcgaa 780atcggtttgg aaaagttctt ggaagacggt gactaccacg ctatcgttac ccacttcggt 840atgttgggtg gtttgcaaca attgccaggt ttggctatcc aaagattgat ggaaaagggt 900tacggtttcg gtggtgaagg tgactggaag actgctgcta tggtcagatt gatgaagatc 960atggctgctg gtgttccagg tgctaagggt acttctttca tggaagacta cacttacaac 1020ttggtcccag gtaaagaagg tatcttgcaa gctcacatgt tggaagtctg tccatctatc 1080gctgaaggtc caatctccat caaggttcaa ccattgtcta tgggtaacag agaagaccca 1140gctagattgg ttttcacctc caagactggt ccagctgtcg ctacctcttt ggttgatttg 1200ggtaacagat tcagattgat catcaacgct gttgactgta agaagtgtga aaaggaaatg 1260ccaaagttgc cagttgctac cgctttctgg actccacaac cagacttggc tactggtgct 1320caagcctgga tcttggctgg tggtgctcac cacaccgctt tctcctacga cttgactgtc 1380gatcaaatgg ttgactgggc tgctgctatg ggtatcgaat ctgttgtcat cgacaaggat 1440accactatca gaaacttcaa gaacgaattg agatggaact ctatctacta cagatga 1497261497DNAunknownSequence from the cow rumen metagenome dataset 26atgatccaaa ctaaggctta caagttctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaagttt tgagacacgt tgctgatcac tctaaggaaa tcgttgaaga attgaacaag 120tccggtatct tgccatacga agttgtctgg aagccagtct tgatcaccaa ccaattgatc 180agacaaactt tcaacgaagc taacgctgac gattcttgtg ctggtgttat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagagttcag aaagccattg 300ttgcacttgc acacccaata caacgaagaa atcccatacg atactatcga catggatttc 360atgaacgaaa accaagctgc tcacggtgac agagaatacg gtcacatcgt ttccagaatg 420ggtatcgaaa gaaaggtcat cgctggttac tggaaggaca acgaagttag atccagaatc 480gcttcctgga tgagaaccgc tgttggtgtc atggaatctt cccacatcag agttatgaga 540gtcgctgata acatgagaaa cgttgctgtc actgaaggtg acaaggttga agctcaaatc 600aagttcggtt gggaagttga cacctaccca gtcaacgaaa tcgctgattc tgttgctact 660gtctctgctt ccgacgtcaa cgctttgttg gacgaatact acgataagta cgaaatcatc 720ttggacggta gagacccaga tgagttcaag aagcacgttg ctgtccaagc tcaaatcgaa 780ttgggtttcg aaagattctt ggaagaaaag aactaccaag ctatcgttac ccacttcggt 840gacttgggtg ctttgggtca attgccaggt ttggctatcc aaagattgat ggaaaagggt 900tacggtttcg gtgctgaagg tgactggaag gttgctgcta tggtcagatt gatgaagatc 960atgacctctg gtatgaagga tgctaagggt acttccatgt tggaagacta cacttacaac 1020ttggttagag gtaaagaagg tatcttggaa gctcacatgt tggaaatctg tccaactatc 1080gctgacggtc caatctctat cagagtcaag ccattgtcta tgggtgacag agaagatcca 1140gctagattgg ttttcacctc taaggaaggt aaaggtgtcg ctacttcctt gatcgacttg 1200ggtaacagat tcagattgat catcaacgaa gttgaatgta agaagaccga aaagccaatg 1260ccaaacttgc cagtcgctac cgcttactgg actccatacc cagacttgta cactggtgct 1320gaagcctgga tcttggctgg tggtgctcac cacaccgctt tctcttacga cttgacttcc 1380ggtcaaatgg ctgattgggc tgaaatgatg ggtatcgaag ctgttatcat cgataagaac 1440accactatcc cagctttcaa gaaggaattg aagttgggtg acgtcttcta cagatga 1497271434DNAunknownSequence from the cow rumen metagenome dataset 27atgaagttct ggttcgttac tggttcccaa ttcttgtacg gtgaagaaac cttgagacaa 60gttgaagaag actctaagaa gatcgttgac ggtttgagat tgccattccc agttgaatac 120aagttgaccg tcaagactga atctgaaatc gaaagaatcg ttaaggaagc taactacgac 180gatgaatgtg ctggtatcat caccttctgt cacactttct ctccatccaa gatgtggatc 240aacggtttgg ctttgttgca aaagccttgg ttgcacttcc acacccaatt caacgaaact 300atcccaaacg aagctatcga catggattac atgaacttgc accaatccgc tcacggtgac 360agagaacacg gtttcatcgg tgctagattg agagttccaa gagctgttgt cgctggttac 420tggaaggacc cagctgtcca agctaagatc ggtgaatggc aaagagctgc tgttggtgtc 480atgttctcca gatccttgaa gatcgtcaga ttcggtgaca acatgagaga agttgctgtc 540accgaaggtg acaagatcga agctcaattg agattgggtt ggcaagttaa caccttcgct 600gttggtgact tggtcgaata catggacgct gtcactgatg ctgaaatcga cgctttgatg 660aaggaatacg ctgaattgta cgaattttct gaagctgaca ccgatactat cagataccaa 720gctagagaag aaatcgctat cgaaaagatt ttggttagag aaggtgctaa ggctttctcc 780aacaccttcg aagacttgca cggtatgaag caattgccag gtttggctac tcaacacttg 840atgcacaagg gttacggttt cggtgctgaa ggtgactgga agaccgctgg tatgactgct 900atcgtcaagg ctatgtaccc agacggtaac acctctttca tggaagatta cacttacgac 960tacgaaagac aattgatctt gggttctcac atgttggaag tttgtccatc catcgctgct 1020gatagaccaa gaatcgaagt ccacaagttg ggtatcggtg gtaaagacgc tccagctaga 1080atcgttttcg aaggtagagc tggttctgct aaggtcttgt ccttgatcga tatcggtggt 1140agattcagat tgatccaaca agacatcgaa tgtgaaaagc cattccaatc tatgccaaac 1200ttgccagttg ctagaaccat gtggagacca gctccatcct tcttggaagg tttggaatgt 1260tggatcatcg ctggtggtgc tcaccacact gttttgtctt acgacatcac cgatgaaact 1320gtcagagatt tcgctagaat catgggtatc gaattggttg tcatcaacaa ggacaccact 1380aaggaaaagt tggaaagaga tatcatgatc ggtgacgtca tctacggtag atga 1434281434DNAunknownSequence from the cow rumen metagenome dataset 28atgaagttct ggttcatcac tggttcccaa ttcttgtacg gtgaagaaac tatcagacaa 60gtcgaagaag attccaagaa gatcgtcgac ggtttgaagt tgccattccc agttgaatac 120aagttgaccg tcaagaagga atctgaaatc gaaagaatcg ttaaggaagc taacttcgac 180gatgaatgtg ctggtatcat caccttctgt cacactttct ctccatccaa gatgtggatc 240aacggtttgg ctatcttgca aaagccttgg ttgcacttcc acacccaatt caacgaaact 300atcccaaacg aagctatcga catggcttac atgaacttgc accaatctgc tcacggtgac 360agagaacacg gtttcatcgg tgctagattg agaatgccaa gagctgttgt cgctggttac 420tggaaggacc cagaagttca agctaagatc gctgaatggc aaagagctgc tgttggtgtc 480atgttctcta agtccttgaa gatcgtcaga ttcggtgaca acatgagaga agttgctgtc 540accgaaggtg acaagatcga agctcaattg aagttgggtt ggcaagtcaa caccttcgct 600gttggtgact tggtcgaata catgaacgct gttactgacg ctgaaatcga tgtcttgatg 660aaggaatacg ctgaattgta cgactacgat aaggctgacg aagaaactat cagataccaa 720gctagagaag aaatcgctat cgaaaagatt ttggttagag aaggtgctaa ggctttctct 780aacaccttcg aagacttgca cggtatgcaa caattgccag gtttggctac tcaacacttg 840atgcacaagg gttacggttt cggtgctgaa ggtgactgga agaccgctgg tatgactgct 900atcgtcaagg ctatgtaccc agacggtaac acctccttca tggaagacta cacttacgat 960tacgaaagaa agttgatctt gggttctcac atgttggaag tttgtccatc catcgctgct 1020gacagaccaa gaatcgaagt ccacccattg ggtatcggtg gtaaagaacc accagctaga 1080atcgttttcg aaggtaaagc tggttctgct aaggtcttgt ccttgatcga catcggtggt 1140agattgagat tgatccaaca agatatcgaa tgtgaaaagc cattccaatc tatgccaaac 1200ttgccagttg ctagaactat gtggagacca gctccatcct tcttggaagg tttggaatgt 1260tggatcatcg ctggtggtgc tcaccacacc gttttgtctt acgacatctc cgatgaaact 1320gtcagagact tcgctagaat catgggtatc gaattggttg tcatcaacaa ggataccact 1380aaggaaaagt tggaaagaga catcatgatc ggtgacatga tctacggtag atga 1434291497DNAunknownSequence from the cow rumen metagenome dataset 29atgtccgaaa tgaagaagta ccaattctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaatgtt tggctcacgt tgctgctcac tctaaggaaa tggtcgaagg tttgaacaag 120tccggtgtct tgccattcga aatcgtttgg aagccaacct tgatcactaa cgaattgatc 180agaaagacct tcaacgaagc taacaacgac ccaaactgtg ctggtgttat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagagttcag aaagccattg 300ttgcacttgc acacccaata caacgaagaa atcccatacg ctactatgga catggatttc 360atgaacgaaa accaagctgc tcacggtgac agagaatacg ctcacatctt gtccagaatg 420agaatcgaaa gaaaggttgt cgttggtttc tggaaggatt ctgaagtcca aaagaagatc 480gcttcctgga tgagaaccgc tatcggtatc atggaatctt cccacatcag agtctgtaga 540gttgctgaca acatgagaaa cgtcgctgtt actgaaggtg acaaggtcga agctcaattg 600aagttcggtt gggaaatcga cgcttaccca gttaacgaaa tcgctgaagc tgtcgctgct 660gtttctgctt ccgacaccaa cgctttggtc gatgaatact actctaagta cgacatctgt 720ttggaaggta gagatccaga agagttcaag aagcacgtcg ctgttcaagc tcaaatcgaa 780atcggtttcg aaagattctt gaaggaaaag aactaccaag ctatcgttac tcacttcggt 840gacttgggtg ctttgaagca attgccaggt ttggctatcc aaagattgat ggaaaagggt 900tacggtttcg gtgctgaagg tgactggaag gtcgctgcta tggttagatt gatgaagatc 960atgtctgctg gtatgaagga cgctaagggt tcttccatgt tggaagatta cacctacaac 1020ttggtcaagg gtaaagaagg tatcatccaa gctcacatgt tggaaatctg tccatctatc 1080tccgacggtc caatccaaat caagtgtcaa ccattgtcta tgggtgacag agaagatcca 1140gctagattgg ttttccaatc taagaccggt gctggtatcg ctacttcctt gatcgacttg 1200ggtaacagat tcagattgat catccaagat gtcgaatgta agaaggttga aaagccattg 1260ccaaagttgc caaccgctat caacttctgg actccacaac cagacttcta caccggtact 1320gaagcctggt tgttggctgg tggtgctcac cacaccgctt tctcttacga catcactgct 1380gaacaaatgg gtgactgggc tgctgctatg ggtatcgaag ctgtcttcat cgacaagaac 1440actaacatca gagacttcaa gaaggatttg atgttgggtg aagttttcta cagatga 1497301464DNAunknownSequence from the cow rumen metagenome dataset 30atgcaaagag aattctggtt catcgtcggt tcccaattct tgtacggtca agatgttttg 60gacactgttg atgctagagc tagagaaatg gctgctgaat tgtctaaggt cttgccatac 120ccattggtct acaaggttac cgctaagact aacaaggaaa tcgctgacac tgttaaggaa 180gctaactaca gagatgaagt catgggtatc gttacctggt gtcacacttt ctctccatcc 240aagatgtgga tcaacggttt ggtcaacttg caaaagccat actgtcactt ggctacccaa 300tacaacagag aattgccaaa cgaagaaatc gacatcgatt tcatgaactt gaaccaagct 360gctcacggtg acagagaaca cggtttcatc gctgctagat tgagaatgcc aagaaaggtc 420atcgctggtt actggcaaga cgaaaaggtt cacaagagat tgtctgattg gatgaaggct 480gctgttggtg ttgacgtttc caagcacatg aaggtcatga gattcggtga caacatgaga

540gaagtcgctg ttaccgaagg tgacaaggtc gaaactcaaa tcaagttggg ttggcaagtt 600aacacttggg ctgtcggtga cttggttaag gaaatgaaca acgttaccga agctgaaatc 660gacgctttgt tcgctgaata cgaagctcaa tacgacatcg ctactgataa cttggctgct 720atcagatacc aagctaagga agaaatcgct atgaagaaga tgttggatag agaaggttgt 780aaggctttct ctaacacctt ccaagacttg tacggtatgg aacaattgcc aggtttggct 840tcccaacact tgatggctca aggttacggt tacggtggtg aaggtgactg gaaggtctct 900gctatgactg ctatcttgaa ggctatgggt gaaaacggta acggtgcttc cgctttcatg 960gaagactaca cctaccactt ggtcgaaggt caagaatact ctttgggtgc tcacatgttg 1020gaagtttgtc catccttggc tgctgacaag ccaagaatcg aaactcacca cttgggtatc 1080ggtatgaacg aaaaggaccc agctagattg gtcttcgaag gtaaagctgg taaaggtatc 1140gttacctctt tgatcgatat gggtggtaga atgagattga tcgtccaaga catcgaagct 1200gttaagccaa tcttgccaat gccaaacttg ccagtcgcta gagttatgtg gagagctatg 1260ccagacttga ccactggtgt tgaatgttgg atcaccgctg gtggtgctca ccacactgtc 1320ttgtctttcg acgttacccc agctatgttg agagactggg ctagaatgat ggatatcgaa 1380tttgtctaca tcactaagga taccactcca gaagaattgg aagaagaatt gttgatcaag 1440gacttggttt ggaagttgaa gtga 1464311500DNAunknownSequence from the human microbiome dataset 31atgttgaaga ctaagaacta ccaattctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaatgtt tggctcacgt tgctgaacac tctaagatca tcgttgacgc tttgaacaag 120tccggtaact tgccatacga agttgtctgg aagccaacca tgatcactaa cgaagttatc 180agaaagacct tcaacgaagc taacactgac gaaaactgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagaatacag aaagccattg 300ttgcacttgc acacccaatt caacagagaa atcccatacg acactatcga catggatttc 360atgaacgaaa accaagctgc tcacggtgac agagaatacg gtcacatctt ctccagattg 420aacatggaaa gaaaggttgt cgctggttac tgggaagacg aagatgttca aaagcaaatc 480ggttcctgga tgagaaccgc tgtcggtgtt gtcgaatctt cccacgttag agtcatgaga 540gttgctgaca acatgagaaa cgttgctgtc actgaaggtg acaaggtcga agctcaaatc 600aagttcggtt gggaagttga cgcttaccca gtcaacgaag ttgtcgaagc tgttaacgct 660gtctctcaag ctgacatcga taccttggtt gaagaatact acgacaagta cgatatcttg 720ttggaaggta gagacgaaaa ggagttcaga gaacacgttg ctgtccaagc tggtatcgaa 780ttgggtttcg aaagattctt ggacgaaaac aactaccaag ctgttgtcac tcacttcggt 840gacttgggtg gtttgaagca attgccaggt ttggctatgc aaagattgat ggaaaagggt 900tacggtttcg gtgctgaagg tgactggaag accgctgcta tggttagagt catgaagatc 960atgactcaag gtatgaagga cgctaagggt acttctttca tggaagatta cacttacaac 1020ttggtttccg gtaaagaagg tgtcttggaa gctcacatgt tggaagtctg tccaaccatc 1080gctgacggta aaatctctat caaggaacaa ccattgtcta tgggtaacag agaagaccca 1140gctagattgg ttttcacctc taagactggt ccagctatcg ctacctcctt gatcgacttg 1200ggtgacagat tcagattgat catcaacgac gtcgattgta agaagactga aaagccaatg 1260ccaaagttgc cagttgctac cgctttctgg actccacaac caaacttgaa ggtcggtact 1320gaagcctgga tcttggctgg tggtgctcac cacaccgctt tctcttacga cttgactgct 1380gaacaaatgg gtgactgggc tgcttgtatg ggtatcgaag ctgtttacat cgacaaggat 1440accactatca gacaattcaa gaacgaattg ttgtggaact ctgtcgctta cagaaagtaa 1500321497DNAunknownSequence from the human microbiome dataset 32atgactggtg ttaagaacta caagttctgg ttctgtactg gttcccaaga tttgtacggt 60gaagaatgtt tggctcacgt cgctgaacac tccagaatca tcgttgaatc tttgaacaga 120tccggtatct tgccatacga agttgtctgg aagccaacct tgatcactaa cgaattgatc 180agaagaacct tcaacgaagc taacgctgac gaagaatgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagagttcag aaagccattg 300atgcacttcc acacccaatt caacagagaa atcccatacg acactatcga catggatttc 360atgaacgaaa accaatccgc tcacggtgac agagaatacg gtcacatggt tactagaatg 420ggtatcgaaa gaaaggttat cgtcggtcac tggtctgacg aaaaggttgt cggtagaatc 480gctggttgga tgagaaccgc tgttggtatc atggaatctt cccacgtcag agttgtcaga 540ttcgctgaca acatgagaaa cgttgctgtc actgaaggtg acaaggttga agctcaagtc 600aagttcggtt gggaagttga cgcttaccca gtcaacgaat tgtgtcaata cgttaaggct 660gtcccaaagg gtgacatcac cgctttggtc gatgaatact actccaagta cactatcttg 720ttggaaggta gagacccaga agagttcaag agacacgttg ctgtccaagc tcaaatcgaa 780gctggtttgg aaagattctt ggttgaaaag gactaccacg ctatcgtcac ccacttcggt 840gacttgggtg aattgcaaca attgccaggt ttggctatcc aaagattgat ggaaaagggt 900tacggtttcg gtggtgaagg tgactggaag actgctgcta tggttagatt gatgaagatc 960atggctcaag gtgtcaagaa cgctaagggt acttctttca tggaagacta cacttacaac 1020ttggttccag gtaaagaagg tatcttggaa gctcacatgt tggaagtttg tccatctatc 1080gctgacggtg aaatctccat caaggtcaac ccattgtcta tgggtgacag agaagatcca 1140gctagattgg ttttcacctc caagactggt cacggtatcg ctacctcttt ggttgacttg 1200ggtactagat tcagattgat catcaacgat gtcgaatgta gaaagaccga aaaggctatg 1260ccaaagttgc cagtcgctac cgctttctgg actccagaac catctttggc tactggtgct 1320gaagcctgga tcttggctgg tggtgctcac cacaccgctt tctcctacga cttgactgct 1380gaacaaatgg gtgactgggc tgaatctatg ggtatcgaag ttgtctacat cgacaaggat 1440accactatca gaggtttgaa gaacgaaatg agatggaacg gtgctgtcta cagataa 1497331497DNAunknownSequence from the human microbiome dataset 33atgatcgctg ttaagaacta caagttctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaatgtt tggctcacgt tgctgaacac tctggtatca tcgttgactc tttgaacaag 120tccggtatct tgccatacga agttgtcttg aagccaacct tgatcactaa cgaattgatc 180agaagaacct tcaacgaagc taacgctgac gaagaatgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagaatacag aaagccattg 300atgcacttcc acacccaatt caaccaagaa atcccatacg actctatcga catggatttc 360atgaacgaaa accaatccgc tcacggtgac agagaatacg gtcacatggt tactagaatg 420ggtatcgaaa gaaaggttat cgtcggtcac tggagagacg aaaaggttgt cggtagaatc 480gctgcttgga tgagaaccgc tgttggtatc atggaatctt cccacgttag agtcgctaga 540ttcgctgaca acatgagaaa cgttgctgtc actgaaggtg acaaggtcga agctcaaatg 600aagttcggtt gggaagttga cgcttaccca gtcaacgaat tggctgaata cgttaaggct 660gtcccaaagg gtgacatcac cgctttggtc gatgaatact actctaagta cactatcttg 720ttggaaggta gagacccaga agagttcaag agacacgttg ctgtccaagc tcaaatcgaa 780gctggtttgg aaaagttctt gttggaaaag gactaccacg ctatcgttac ccacttcggt 840gacttgggtg aattgcaaca attgccaggt ttggctatcc aaagattgat ggaaaagggt 900tacggtttcg gtgctgaagg tgactggaag accgctgcta tggtcagatt gatgaagatc 960atgactcaag gtatgaagga cgctaagggt acttctttca tggaagatta cacttacaac 1020ttggttccag gtaaagaagg tatcttggaa gctcacatgt tggaagtctg tccaactatc 1080gctgacggtg aaatctctat caaggcttgt ccattgtcta tgggtgacag agaagatcca 1140gctagattgg ttttcacctc taagactggt cacggtatcg ctgcttcctt ggttgacttg 1200ggtactagat tcagattgat catcaacgat gtcgaatgta agaagactga aaagccaatg 1260ccaaagttgc cagtcgctac cgctttctgg actccagaac caaacttggc taccggtgct 1320gaatcttgga tcttggctgg tggtgctcac cacaccgctt tctcctacga cttgactgct 1380gaacaaatgg gtgactgggc tgatgctatg ggtatcgaaa ctgtttacat cgacaaggat 1440accactatca gaggtttgaa gaacgaattg agatggaacg ctgctgctta cagataa 1497341500DNAunknownSequence from the human microbiome dataset 34atgttgaaga agaaggaata caagttctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaatgtt tggctcacgt tgctgaacac gctaagatca tcgtcgaaaa gttgaacgaa 120tccggtgttt tgccatacga agttgtctgg aagccaacct tgatcactaa cgaattgatc 180agaaagacct tcaacgaagc taacatcgac gatgaatgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagagttcag aaagccattg 300ttgcacttgc acacccaatt caacatggaa atcccatacg acactatcga catggatttc 360atgaacgaaa accaatctgc tcacggtggt agagaatttg gtcacatctt cactagattg 420ggtatcgaaa gaaaggttgt cgttggtcac tggtccgacg aaaaggttca agaaaagatc 480gcttcttgga tgagaaccgc tgtcggtgtt atcgaatctt cccacgtcag agttatgaga 540gtcgctgaca acatgagaaa cgtcgctgtt actgaaggtg acaaggttga agctcaaatc 600aagttcggtt gggaagttga cgcttaccca gttaacgaaa tcgctgaatc tgttgacgct 660gtttccgctg ctgatgtcaa caccttggtt gaagaatact acgacaagta cgaaatcttg 720ttggaaggta gagatccaga agagttcaga aagcacgtcg ctgttcaagc tcaaatcgaa 780ttgggtttcg aaagattctt ggaagaaaag aactaccaag ctatcgtcac tcacttcggt 840gacttgggtg ttttgaagca attgccaggt ttggctatcc aaagattgat gcaaaagggt 900tacggtttcg gtgctgaagg tgactggaag accgctgcta tggtcagaat catgaagatc 960atgactgaag gtatgaagga cgctaagggt acttctatgt tggaagatta cacttacaac 1020ttcgttccag gtaaagaagg tatcttgcaa gctcacatgt tggaaatctg tccatctatc 1080gctgacggtc caatctccat caaggtcaac ccattgtcta tgggtgacag agaagatcca 1140gctagattgg ttttcacctc caaggaaggt aaaggtatcg ctacttcttt gatcgacttg 1200ggtgacagat tcagattgat catcaacacc gttgactgta agaagaacga aaagccaatg 1260ccaaagttgc cagttgctac caacttctgg actccagaac cagacttggc tactggtgct 1320gaagcctgga tcttgtgtgg tggtgctcac cacaccgctt tctcttacga catcactgct 1380gaacaaatgg gtgactgggc tgctatgatg ggtatcgaag ctgtctacat cgacaaggat 1440accactatca gaaacttgaa gaacgaattg agatggaacg aattggcttt cagaaagtaa 1500351497DNAunknownSequence from the human microbiome dataset 35atgaaggctg ctaaggatta caagttctgg ttctgtactg gttctcaaga tttgtacggt 60gacgaatgtt tggctcacgt tgctgaacac tccagaatca tcgttgacgc tttgaacaag 120tccggtgttt tgccatacga aatcgtctgg aagccaacct tgatcactaa cgaattgatc 180agaaagacct tcaacgaagc taacgctgac gaaaactgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttgc aagagttcag aaagccattg 300ttgcacttcc acacccaatt caacagagaa atcccatacg acactatcga catggatttc 360atgaacgaaa accaagctgc tcacggtgac agagaatacg gtcacatcgt ttccagaatg 420ggtatcgaaa gaaagatcat cgttggttac tgggaagaca gagatgtcca agaaaagatc 480gcttcctgga tgttgaccgc tatcggtatc atggaatctt cccacatcag agtctgtaga 540atcgctgaca acatgagaaa cgttgctgtc actgaaggtg acaaggttga agctcaaatc 600aagttcggtt gggaaatcga cgcttaccca gtcaacgaaa tcgctgaata cgttgctgct 660gtcccacaag gtgaaatcaa cgctttggtt gaagaatact actctaagta cgacatcatc 720ttggaaggta gagatccaca agagttcaga gaacacgttg ctgtccaagc tggtatcgaa 780atcggtttcg aaaagttctt ggaagaaaag aactaccaag ctatcgtcac tcacttcggt 840gacttgggtt ctttgaagca attgccaggt ttggctatcc aaagattgat ggaaaagggt 900tacggtttcg gtggtgaagg tgactggaag accgctgcta tggttagatt gatgaagatc 960atgactgctg gtgtcaagaa cccaaagggt acttctttca tggaagacta cacttacaac 1020ttggttccag gtaaagaagg tgtcttggaa gctcacatgt tggaagtttg tccatctgtc 1080gctgatggtc caatcggtat caaggtttgt ccattgtcta tgggtgacag agaagatcca 1140gctagattgg tctacacctc taagactggt ccagctatcg ctacctcctt gatcgacttg 1200ggtaacagat tcagattgat catcaacgaa gttgaatgta agaaggtcga aaagccaatg 1260ccaaagttgc cagttgctac cgctttctgg actccatacc cagacttgaa gactggtgct 1320gaagcctgga tcttggctgg tggtgctcac cacaccgctt tctcttacga cttgactgct 1380gaacaaatgg gtgactgggc tgctgctatg ggtatcgaag ctgtttacat cgacaaggat 1440accactatca gaaacttcaa gagagacttg caattgggta acatcgtcta cagataa 1497361497DNAunknownSequence from the human microbiome dataset 36atggttactg gtagaaacta caagttctgg ttctgtactg gttcccaaga tttgtacggt 60gacgaatgtt tgagaaaggt tgctgaacac tccagaatca tcgttgaaga attgaacaag 120tccggtgttt tgccattcga attggtctgg aagccaacct tgatcactaa cgaattgatc 180agaaagacct tcaacgaagc taacgctgac gatgaatgtg ctggtgtcat cacctggatg 240cacactttct ctccagctaa gtcttggatc ttgggtttga aggaatacag aaagccattg 300tgtcacttgc acacccaatt caaccaagaa atcccatacg acactatcga catggatttc 360atgaacgaaa accaatctgc tcacggtggt agagaatacg gtcacatcgt tactagaatg 420ggtatcgaaa gaaaggttat cgtcggtcac tgggctgaca agaaggttca agaaagattg 480gcttcctgga tgagaaccgc tgtcggtatc atggaatctt cccacatcag agtttgtaga 540gtcgctgaca acatgagaaa cgttgctgtc actgaaggtg acaaggttga agctcaaatc 600aagttcggtt gggaagttga cgcttaccca gttaacgaag tctgtgacta cgttaaggat 660gtctctaagg gtgacatcga tgttttggtc gaagaatact acaacaagta cgacatcttg 720ttcgaaggta gagatccaga agagttcaag agacacgttg ctgtccaagc tgctatcgaa 780atcggtttcg aaagattctt ggaagaaaag aactaccaag ctgttgtcac ccacttcggt 840gacttgggtg gtttgcaaca attgccaggt ttggctatgc aaagattgat ggaaaagggt 900tacggtttcg gtgctgaagg tgactggaag accgctgcta tggttagatt gatgaagatc 960atgactgctg gtgtcaagga cgctaagggt acttctttca tggaagatta cacttacaac 1020ttggttccag gtaaagaagg tatcttgcaa tcccacatgt tggaagtttg tccaaccatc 1080gctgacggta aaatcggtat caaggtctgt ccattgtcta tgggtgacag agaagatcca 1140gctagattgt tcacctctaa gactggtcca gctgttgcta cttccttggt tgacttgggt 1200gacagattca gattgatcat caacgacgtt gattgtaaga aggtcgaaaa gccaatgcca 1260aagttgccag tcggttctgc tttctggacc ccacaaccag acttggctac tggtgctgaa 1320gcctggatct tggctggtgg tgctcaccac accgctttct cctacgactt gactgctgaa 1380caaatgggtg actgggctgc tgctatgggt atcgaagctg tttacatcga caaggatacc 1440actatcagaa acttcaagaa cgaattgaga tggaacgaag tcgctttcag aaagtaa 1497371476DNAunknownSequence from the cow rumen metagenome dataset 37atggaagaca tcatgaagag agaattttgg ttcatcgttg gttcccaatt cttgtacggt 60caagacgttt tggacactgt tgacgctaga gctaaggaaa tggctgctga attgtctaag 120gtcttgccat acccattggt ctacaaggtt accgctaaga ctaacaagga aatcactgac 180gtcatcaagg aagctaacta cagagatgaa tgtgctggta tcgttacctg gtgtcacact 240ttctctccat ccaagatgtg gatcaacggt ttggctaact tgcaaaagcc atactgtcac 300ttggctaccc aatacaacaa ggaaatccca aacgacgaaa tcgacatgga tttcatgaac 360ttgaaccaag ctgctcacgg tgacagagaa cacggtttca tcgctgctag attgagattg 420ccaagaaagg ttatcgctgg tttctggcaa gacgaaaaga tccacaagag attgtctgat 480tggatgagag ctgctgttgg tgtcgctgtt tccaagaaga tgaaggtcat gagattcggt 540gacaacatga gagaagtcgc tgttactgaa ggtgacaagg tcgaagttca aactaagttg 600ggttggcaag ttaacacctg ggctgtcggt gacttggtta aggaaatggg taaagtcacc 660gaagctgaaa tcgatgcttt ggttgctgaa tacgaagcta actacgacat cgctaccgat 720aacactgctg ctatcagata ccaagctaga gaagaaatcg ctatgaagaa gatgttggac 780agagaaggtt gtagagcttt caccaacact ttccaagatt tgtacggtat ggaacaattg 840ccaggtttgg cttctcaaca cttgatggct caaggttacg gttacggtgg tgaaggtgac 900tggaaggtct ctgctatgac tgctatcttg aaggctatgg gtgaaaacgg taacggtgct 960tccggtttca tggaagacta cacctaccac ttggtcgaag gtcaagaata ctctttgggt 1020gctcacatgt tggaagtttg tccatccttg gctgctgaca agccaagaat cgaaactcac 1080cacttgggta tcggtatgaa cgaaaaggac ccagctagat tggttttcga aggtaaagct 1140ggtaaaggta tcgttgtctc tttgatcgac atgggtggta gattgagatt gatcgtccaa 1200gatatcgaag ctgttaagcc aatcttgcca atgccaaact tgccagtcgc tagagttatg 1260tggagagcta tgccagactt gaccactggt gtcgaatgtt ggatcaccgc tggtggtgct 1320caccacactg tcttgtccta cgacgttacc gctgaacaaa tgagagactg ggctagaatg 1380atggatatcg aatttgttca catcaccaag gacactaccc cagaaaagtt ggaagaagaa 1440ttgttggtta aggatttggt ttggaagttg aagtaa 1476381467DNAunknownSequence from the cow rumen metagenome dataset 38atgtctaagg aattttggtt cgtcgtcggt tcccaagatt tgtacggtga agaagttttg 60aagatcgtcg ctgaaagagc tgctgaaatg gctgcttggt tgtctgaaaa gttgccatac 120ccattgatct acaaggtcac tgctatgtct tccaaccaaa tcacctccgt tatgaaggaa 180gctaacttcg acgataactg tttgggtgtt gtcacctggt gtcacacttt ctctccatcc 240aagatgtggt tgactggttt ggacttgttg caaaagcctt ggtgtcactt cgctacccaa 300tacaacttgg aaatcccaaa cgaagaaatc gacatggatt tcatgaactt gaaccaagct 360gctcacggtg acagagaaca cggtttcatc ggtgctagat tgagaaaggc tagaaaggtt 420gtcgctggtt actggaagga cgaaaaggtc atcgctagat tggctgaatt tcaaaaggtt 480gctgtcggtg ttgacgcttc taagcacatg aaggttatga gattcggtga caacatgaga 540gatgtcgctg ttactgaagg tgacaaggtc gaagttcaaa agaagttggg ttgggaagtc 600aacacttggg ctgtcggtga cttggttaag gaaatgaacg ctgtcaccga tgaagaagtt 660gaagccttgt tcaacgaata caaggcttct tacgacatca acactgataa catctacgct 720atcaagtacc aagctagaga agaaatcgct atcaagaaga tgatggacag aaacggttgt 780aaggctttct ccaacacctt ccaagatttg tacggtatgg aacaattgcc aggtttggct 840tctcaacact tgatgtcctt gggttacggt tacggtggtg aaggtgactg gaaggtttct 900gctatgactg ctatcttgaa ggctatgggt gaaaacggta acggtgcttc cgctttcatg 960gaagactaca cctaccactt ggtcaagggt cacgaatact ctttgggtgc tcacatgttg 1020gaagtttgtc catccttggc tgctgacaag ccaagaatcg aaacccacca cttgggtatc 1080ggtatgaacg aaaaggaccc agctagattg gtcttcgaag gtaaagaagg tagaggtatc 1140gttgcttctt tgatcgacat gggtggtaga ttgagattga tcgtccaaga tatcgaagct 1200gttaagccaa tcatgccaat gccaaacttg ccagtcgcta gagttatgtg gagagctttg 1260ccagacttga ctgatggtgt cgaatgttgg atcaccgctg gtggtgctca ccacactgtc 1320ttgtcttacg acgttacccc agaaatgatg agagacttcg ctaagttcat ggatatcgaa 1380tttgttcaca tcgacaagga taccaccgtt gaaaagttgg aagatgaatt gatggttaag 1440gatttggttt ggaagatgaa gggttaa 1467391467DNAunknownSequence from the cow rumen metagenome dataset 39atgggtaaca agaagaactt ctggttcgtc gtcggttctc aattcttgta cggtaacgaa 60gttttggaaa ctgtcgctgc tagagctcaa gaaatggctg aaaagatgtc taagtccttg 120ccatacgaat tgaagttcaa gggtatcgtc aagacctggg acgaagctac tcaatacgct 180aaggaagcta acttcgacga taactgttgt ggtgttatca cctggtgtca cactttctct 240ccatccaaga tgtggatcga agccttcaga ttgttgcaaa agccattgtt gcacttcgct 300acccaataca acagatacat cccagacaag gaaatcgaca tggatttcat gaacttgaac 360caagctgctc acggtgacag agaacacggt ttcatcatcg ctagaatgag attgcaacaa 420aagatcgtca ccggtttctg ggaagaccaa ccagttttgg atgaaatcgg tacttggatg 480agagctgctg tcgcttacga cttctccaga aacttgagag ttatgagatt cggtgacaac 540atgagagaag tcgctgttac tgaaggtgac aaggtcgaag ctcaaatcaa gttcggttgg 600caagttaaca cctggccagt cggtaaattg gttgaagaaa tcggtaaagt cactgaagaa 660gaagttgacg aattgttgaa ggtctacacc gatacttacg aattggctac cgacgatatc 720gaaactatca gataccaagc tagagaagaa atcgctatga agaagatgat gaccgctgaa 780ggtgctaacg ctttcgttaa cactttccaa gacttgatcg gtatgaagca attgccaggt 840atcgcttctc aacacttgat ggctcaaggt tacggttacg gtgctgaagg tgactggaag 900ttgtctgctt tggtctccat cgttaagaag atgaccgaag gtatgaccgg tggtacttct 960ttcatggaag actacactta ccacttggac ccaaacgctg aatacgcttt gggtgctcac 1020atgttggaag tctgtccatc catcgctgct gacaagccaa gaatcgaagt tcacccattg 1080ggtatcggtg acagagaaga tccagctaga ttggtcttcg aaggtcacga aggtgacgct 1140gttgtcgtta ccttgatcga tatgggtgaa agattcagaa tgttggtcca agacatccac 1200tgtgttaagc caatctacga aatgccaaac ttgccagtcg ctagagttat gtgggaaggt 1260aaaccatctt tgaacgaagg tttgaagatg tggttgatgg ctggtggtgc tcaccactct 1320gtcttgtcct acgacgctac cccagaaatg ttgaaggact tggctagaat gatggatatc 1380gaatttgttc acatcactgc tgactccaag ccagaagaat ttgaaaagga cttgttcttc 1440gctgatttgg cttggaagtt gaagtaa 1467401413DNAunknownSequence from the cow rumen metagenome dataset

40atgaagaaga tttacttcat cactggttcc caagacttgt acggtgaaga cgttttgaag 60actgtcgcta aggactccca agaaatggtt aacttcttgg acgaacaagt cggtgaaaga 120gctgaaatcg aatttttggg tgttgtcaga gactctgaaa tctgtttgga tttcatcttg 180aaggctaact tcgacaagga atgtatcggt atcatcacct ggatgcacac tttctcccca 240gctaagatgt ggatcagagg tttgaaggtt ttgcacaagc caatgttgca cttgcacacc 300caatacaacg aaaagttgcc atacgactct atcgacatgg atttcatgaa cttgaaccaa 360gctgctcacg gtgacagaga atttggtttc atcgctgcta gaatgaacat caagcaacac 420gttttgtccg gttactacaa gaacaaggac ttcatcgaag gtgttaagca atacatcgac 480gtctgtttgt ctatcgatgc tgctaagtac ttgagagtcg ctatgttcgg ttccaacatg 540agagacgttg ctgtcactga cggtgacaga gttcaatctg aaatcgactt cggttggaac 600gtcaactact acggtatcgg tgacttggtt gatatcatca acaaggtcaa ggacgaagaa 660atcgatgctc aattcgaaga atacaagaag agatacacca tcaacaccac taacatcgaa 720gctatcaagg aacaagccaa gtacgaagtt gctttgaaga agttcatcaa gaaggaaaac 780gtccaagcct tcaccgacaa cttccaagat ttgcacggtt tgaagcaatt gccaggtttg 840gctgttcaag acttgatgca agaaggtatc tctttcggtc cagaaggtga ctacaagacc 900ccagctttgt tggctacctt gttgccaatg actaagtaca gaaagggtgc taccggtttc 960atcgaagact acacttacga tttgatcgaa ggtaaagaaa tcgaattggg ttctcacatg 1020ttggaagttc caccatcttt cgctacttcc aagccagaaa tccaagtcag accattgtct 1080atcggtgaca aggctgctcc agctagattg gttttcgatt ccatcgaagg tgaaggtttg 1140caaatcacta tggttgacat gggtactcac ttcagaatca tcgctgctaa gatccaattg 1200gttaagcaac cagctccaat gccaaagttg ccagtcgcta gaatcatgtg gaagcacatc 1260ccaaacttca agatttctac cgaagcctgg atgttgtacg gtggtggtca ccactccgtc 1320atcaccactg ctttgactat cgaagacatc aagttgttcg ctaagttgac tggtactgaa 1380ttgtgtgtta tcgatgaaaa cactaagatt taa 141341496PRTBacillus subtilis 41Met Leu Gln Thr Lys Asp Tyr Glu Phe Trp Phe Val Thr Gly Ser Gln1 5 10 15His Leu Tyr Gly Glu Glu Thr Leu Glu Leu Val Asp Gln His Ala Lys 20 25 30Ser Ile Cys Glu Gly Leu Ser Gly Val Ser Ser Arg Tyr Lys Ile Thr 35 40 45His Lys Pro Val Val Thr Ser Ser Glu Thr Ile Arg Gln Leu Leu Arg 50 55 60Glu Ala Glu Tyr Ser Glu Thr Cys Ala Gly Ile Ile Thr Trp Met His65 70 75 80Thr Phe Ser Pro Ala Lys Met Trp Ile Glu Gly Leu Ser Ser Tyr Gln 85 90 95Lys Pro Leu Met His Leu His Thr Gln Tyr Asn Arg Asp Ile Pro Trp 100 105 110Gly Thr Ile Asp Met Asp Phe Met Asn Ser Asn Gln Ser Ala His Gly 115 120 125Asp Arg Glu Tyr Gly Tyr Ile Asn Ser Arg Met Gly Leu Ser Arg Lys 130 135 140Val Val Ala Gly Tyr Trp Asp Asp Glu Glu Val Lys Lys Glu Ile Ser145 150 155 160Gln Trp Met Asp Thr Ala Ala Ala Leu Asn Glu Ser Arg His Ile Lys 165 170 175Val Ala Arg Phe Gly Asp Asn Met Arg His Val Ala Val Thr Asp Gly 180 185 190Asp Lys Val Gly Ala His Ile Gln Phe Gly Trp Gln Val Asp Gly Tyr 195 200 205Gly Ile Gly Asp Leu Val Glu Val Met Asn Arg Ile Thr Asp Asp Glu 210 215 220Val Asp Thr Leu Tyr Ala Glu Tyr Asp Arg Leu Tyr Val Ile Ser Glu225 230 235 240Glu Thr Lys Arg Asp Glu Ala Lys Val Ala Ser Ile Lys Glu Gln Ala 245 250 255Lys Ile Glu Leu Gly Leu Thr Thr Phe Leu Glu Gln Gly Gly Tyr Ser 260 265 270Ala Phe Thr Thr Ser Phe Glu Val Leu His Gly Met Lys Gln Leu Pro 275 280 285Gly Leu Ala Val Gln Arg Leu Met Glu Lys Gly Tyr Gly Phe Ala Gly 290 295 300Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Met Met Lys Ile Met305 310 315 320Ser Gln Gly Lys Arg Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Phe 325 330 335Glu Pro Gly Asn Glu Met Ile Leu Gly Ser His Met Leu Glu Val Cys 340 345 350Pro Thr Val Ala Leu Asp Gln Pro Lys Ile Glu Val His Pro Leu Ser 355 360 365Ile Gly Gly Lys Glu Asp Pro Ala Arg Phe Val Phe Asn Gly Ile Ser 370 375 380Gly Ser Ala Ile Gln Ala Ser Leu Val Asp Ile Gly Gly Arg Phe Arg385 390 395 400Leu Val Leu Asn Glu Val Asn Gly Gln Glu Ile Glu Lys Asp Met Pro 405 410 415Asn Leu Pro Val Ala Arg Val Leu Trp Lys Pro Glu Pro Ser Leu Lys 420 425 430Thr Ala Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala His His Thr Cys 435 440 445Leu Ser Tyr Glu Leu Thr Val Glu Gln Met Leu Asp Trp Ala Glu Met 450 455 460Ala Gly Ile Glu Ser Val Leu Ile Ser Arg Asp Thr Thr Ile His Lys465 470 475 480Leu Lys His Glu Leu Lys Trp Asn Glu Ala Leu Tyr Arg Leu Gln Lys 485 490 495421491DNAartificial sequenceCodon-optimized coding region of B. subtilis AI. 42atgttgcaaa ctaaggatta cgaattctgg ttcgttactg gttctcaaca cttgtacggt 60gaagaaactt tggaattggt cgatcaacac gctaagtcta tctgtgaagg tttgtccggt 120gtctcttcca gatacaagat cacccacaag ccagttgtca cctcttccga aactatcaga 180caattgttga gagaagctga atactctgaa acttgtgctg gtatcatcac ctggatgcac 240actttctctc cagctaagat gtggatcgaa ggtttgtctt cctaccaaaa gccattgatg 300cacttgcaca cccaatacaa cagagacatc ccttggggta ctatcgacat ggatttcatg 360aactctaacc aatccgctca cggtgacaga gaatacggtt acatcaactc cagaatgggt 420ttgtccagaa aggttgtcgc tggttactgg gacgatgaag aagtcaagaa ggaaatctct 480caatggatgg acaccgctgc tgctttgaac gaatccagac acatcaaggt tgctagattc 540ggtgacaaca tgagacacgt tgctgtcact gacggtgaca aggttggtgc tcacatccaa 600ttcggttggc aagttgacgg ttacggtatc ggtgacttgg ttgaagtcat gaacagaatc 660accgacgatg aagttgacac tttgtacgct gaatacgata gattgtacgt catctctgaa 720gaaaccaaga gagacgaagc taaggttgct tccatcaagg aacaagctaa gatcgaattg 780ggtttgacca ctttcttgga acaaggtggt tactctgctt tcaccacttc cttcgaagtc 840ttgcacggta tgaagcaatt gccaggtttg gctgttcaaa gattgatgga aaagggttac 900ggtttcgctg gtgaaggtga ctggaagacc gctgctttgg tcagaatgat gaagatcatg 960tctcaaggta aaagaacctc cttcatggaa gactacactt accacttcga accaggtaac 1020gaaatgatct tgggttctca catgttggaa gtttgtccaa ctgtcgcttt ggaccaacca 1080aagatcgaag ttcacccatt gtctatcggt ggtaaagaag atccagctag attcgtcttc 1140aacggtatct ctggttccgc tatccaagcc tctttggttg acatcggtgg tagattcaga 1200ttggttttga acgaagtcaa cggtcaagaa atcgaaaagg acatgccaaa cttgccagtt 1260gctagagtct tgtggaagcc agaaccatct ttgaagactg ctgctgaagc ctggatcttg 1320gctggtggtg ctcaccacac ctgtttgtct tacgaattga ctgtcgaaca aatgttggac 1380tgggctgaaa tggctggtat cgaatctgtt ttgatctcca gagataccac tatccacaag 1440ttgaagcacg aattgaagtg gaacgaagcc ttgtacagat tgcaaaagta a 14914316404DNAartificial sequenceconstructed plasmid 43aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 60gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 120tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 180agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 240gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 300gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 360aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt aggcgcctac 420ttctaggggg cctatcaagt aaattactcc tggtacactg aagtatataa gggatataga 480agcaaatagt tgtcagtgca atccttcaag acgattggga aaatactgta atataaatcg 540taaaggaaaa ttggaaattt tttaaagatg tcttcactgg ttactcttaa taacggtctg 600aaaatgcccc tagtcggctt agggtgctgg aaaattgaca aaaaagtctg tgcgaatcaa 660atttatgaag ctatcaaatt aggctaccgt ttattcgatg gtgcttgcga ctacggcaac 720gaaaaggaag ttggtgaagg tatcaggaaa gccatctccg aaggtcttgt ttctagaaag 780gatatatttg ttgtttcaaa gttatggaac aattttcacc atcctgatca tgtaaaatta 840gctttaaaga agaccttaag cgatatggga cttgattatt tagacctgta ttatattcac 900ttcccaatcg ccttcaaata tgttccattt gaagagaaat accctccagg attctatacg 960ggcgcagaag gattctatac gggcgcagaa ctagtgatct cgaggttcca gagctcggat 1020ccaccacagg tgttgtcctc tgaggacata aaatacacac cgagattcat caactcattg 1080ctggagttag catatctaca attgggtgaa atggggagcg atttgcaggc atttgctcgg 1140catgccggta gaggtgtggt caataagagc gacctcatgc tatacctgag aaagcaacct 1200gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact 1260ttaaaatttg tatacactta ttttttttat aacttattta ataataaaaa tcataaatca 1320taagaaattc gcttactcat cccgggttag atgagagtct tttccagttc gcttaagggg 1380acaatcttgg aattatagcg atcccaattt tcattatcca catcggatat gctttccatt 1440acatgccatg gaaaattgtc attcagaaat ttatcaaaag gaactgcaat tttattagag 1500tcatataaca atgaccacat ggccttataa caaccaccaa gggcacatga gtttggtgtt 1560tctagcctaa aattaccctt tgtagcacca atgacttgag caaacttctt cacaatagca 1620tcgtttttag aagccccacc tacaaaaaaa gtcctttctg gccttttatt taggtagtcc 1680cgcagcggag attcatcgta atcaaacttc acgattgtat cttcgttcag tctctgttgt 1740gagcttgcgt ttgaatccga aagcagggga gatattctta ccctgcaact taaagcctgt 1800gattctacaa tatttttggc atcgtgcctc ttgtctttga acttggccac ctctctttca 1860atcatacccg tttttggatt gaagataacc cttttgttta tggcttttac gctaggaacg 1920atctccccca gaggaaaata tacacctaat tcattttcac tactttctga gtcatctagc 1980acagcttgat taaaaagagt ccaatcgtta gtcttctcat aattattttc ccgttctttg 2040tttaactcgt ctcttatcct ctcccttgcc aaagaaccat tacaataaca aatcataccc 2100atataatggt ttggcagagt tggatgaatg aaaagatgat agttcggaga ggggtgatac 2160ttatcggtga ccagaagaac tgtagtactt gttcctaggg aaacgagaac gtcattcttc 2220cgcaggggta aagaacatat agtggctaaa ttatccccag tcatgggaga gaccttgcag 2280tttgtattga aaccgtactt ctcaataaaa tatttacaga tggtacccgc tatcaaattt 2340ttcatgggtg ctctcattaa tttttgtctg atagttttat ccttagaaga actatcaatt 2400agatgtagta gctcatcact gaattttctt tcacgtatat cataaaggtt cataccacag 2460gcatctgcct cctctaattc aacaagatgg cccactaaga tagaagtcaa aaaattagac 2520actaaagaaa tggtctttgt tttttcgtaa gcttctggtt ctaattgtgc aattttcaga 2580atttgaggac cagtaaatct aaaatgggct ctggaccctg ttaattgagc cattttttca 2640ggcccaccta tgcactcttc aaactcttga cattgctttg cagtactgtg gtcttgccaa 2700ttgggggcgg tttgccttgc aaatgctaca gagctcacgt agtgcaataa atctttttcc 2760ggtttcttat tcaattgctc taacagagat tcggcttggg aggaccagta gacagacccg 2820tgctgctggc aggaccctga gacggccata actttgttca atggaaattt agcctcgcga 2880tatttcgaga gaaccagatc tagagcctct aaccacatgg ctacgggaca ttcgatagtg 2940tcgccgtgta tatagacacc cttctttgtg tgataatgcg gaagatcctt ttcaaattcc 3000actgtttctg aatggacaat ttttaggtcc tggttaatgg cgagacattt cagttgttgg 3060gtcgaaagat caaacccaag atagtatgag tctaaagaca ttgtgttgga aacctctctt 3120gtctgtctct gaattactga acacaacata ctagtcgtac ggttttattt tttacttata 3180ttgctggtag ggtaaaaaaa tataactcct aggaataggt tgtctatatg tttttgtctt 3240gcttctataa ttgtaacaaa caaggaaagg gaaaatactg ggtgtaaaag ccattgagtc 3300aagttaggtc atccctttta tacaaaattt ttcaattttt tttccaagat tcttgtacga 3360ttaattattt tttttttgcg tcctacagcg tgatgaaaat ttccgcctgc tgcaagatga 3420gcgggaacgg gcgaaatgtg cacgcgcaca acttacgaaa cgcggatgag tcactgacag 3480ccaccgcaga ggttctgact cctactgagc tctattggag gtggcagaac cggtaccgga 3540ggagaccgct ataaccggtt tgaatttatt gtcacagtgt cacatcagcg gcaactcaga 3600agtttgacag caagcaagtt catcattcga actagcctta ttgttttagt tcagtgacag 3660cgaactgccg tactcgatgc tttatttctc acggtagagc ggaagaacag ataggggcag 3720cgtgagaaga gttagaaagt aaatttttat cacgtctgaa gtattcttat tcataggaaa 3780ttttgcaagg ttttttagct caataacggg ctaagttata taaggtgttc acgcgatttt 3840cttgttatgt atacctcttc tggcgcgcct ctttttatta accttaattt ttattttaga 3900ttcctgactt caactcaaga cgcacagata ttataacatc tgcataatag gcatttgcaa 3960gaattactcg tgagtaagga aagagtgagg aactatcgca tacctgcatt taaagatgcc 4020gatttgggcg cgaatccttt attttggctt caccctcata ctattatcag ggccagaaaa 4080aggaagtgtt tccctccttc ttgaattgat gttaccctca taaagcacgt ggcctcttat 4140cgagaaagaa attaccgtcg ctcgtgattt gtttgcaaaa agaacaaaac tgaaaaaacc 4200cagacacgct cgacttcctg tcttcctatt gattgcagct tccaatttcg tcacacaaca 4260aggtcctagc gacggctcac aggttttgta acaagcaatc gaaggttctg gaatggcggg 4320aaagggttta gtaccacatg ctatgatgcc cactgtgatc tccagagcaa agttcgttcg 4380atcgtactgt tactctctct ctttcaaaca gaattgtccg aatcgtgtga caacaacagc 4440ctgttctcac acactctttt cttctaacca agggggtggt ttagtttagt agaacctcgt 4500gaaacttaca tttacatata tataaacttg cataaattgg tcaatgcaag aaatacatat 4560ttggtctttt ctaattcgta gtttttcaag ttcttagatg ctttcttttt ctctttttta 4620cagatcatca aggaagtaat tatctacttt ttacaacaaa tataaaacac gtacgactag 4680tatgactcaa ttcactgaca ttgataagtt ggccgtctcc accataagaa ttttggctgt 4740ggacaccgta tccaaggcca actcaggtca cccaggtgct ccattgggta tggcaccagc 4800tgcacacgtt ctatggagtc aaatgcgcat gaacccaacc aacccagact ggatcaacag 4860agatagattt gtcttgtcta acggtcacgc ggtcgctttg ttgtattcta tgctacattt 4920gactggttac gatctgtcta ttgaagactt gaaacagttc agacagttgg gttccagaac 4980accaggtcat cctgaatttg agttgccagg tgttgaagtt actaccggtc cattaggtca 5040aggtatctcc aacgctgttg gtatggccat ggctcaagct aacctggctg ccacttacaa 5100caagccgggc tttaccttgt ctgacaacta cacctatgtt ttcttgggtg acggttgttt 5160gcaagaaggt atttcttcag aagcttcctc cttggctggt catttgaaat tgggtaactt 5220gattgccatc tacgatgaca acaagatcac tatcgatggt gctaccagta tctcattcga 5280tgaagatgtt gctaagagat acgaagccta cggttgggaa gttttgtacg tagaaaatgg 5340taacgaagat ctagccggta ttgccaaggc tattgctcaa gctaagttat ccaaggacaa 5400accaactttg atcaaaatga ccacaaccat tggttacggt tccttgcatg ccggctctca 5460ctctgtgcac ggtgccccat tgaaagcaga tgatgttaaa caactaaaga gcaaattcgg 5520tttcaaccca gacaagtcct ttgttgttcc acaagaagtt tacgaccact accaaaagac 5580aattttaaag ccaggtgtcg aagccaacaa caagtggaac aagttgttca gcgaatacca 5640aaagaaattc ccagaattag gtgctgaatt ggctagaaga ttgagcggcc aactacccgc 5700aaattgggaa tctaagttgc caacttacac cgccaaggac tctgccgtgg ccactagaaa 5760attatcagaa actgttcttg aggatgttta caatcaattg ccagagttga ttggtggttc 5820tgccgattta acaccttcta acttgaccag atggaaggaa gcccttgact tccaacctcc 5880ttcttccggt tcaggtaact actctggtag atacattagg tacggtatta gagaacacgc 5940tatgggtgcc ataatgaacg gtatttcagc tttcggtgcc aactacaaac catacggtgg 6000tactttcttg aacttcgttt cttatgctgc tggtgccgtt agattgtccg ctttgtctgg 6060ccacccagtt atttgggttg ctacacatga ctctatcggt gtcggtgaag atggtccaac 6120acatcaacct attgaaactt tagcacactt cagatcccta ccaaacattc aagtttggag 6180accagctgat ggtaacgaag tttctgccgc ctacaagaac tctttagaat ccaagcatac 6240tccaagtatc attgctttgt ccagacaaaa cttgccacaa ttggaaggta gctctattga 6300aagcgcttct aagggtggtt acgtactaca agatgttgct aacccagata ttattttagt 6360ggctactggt tccgaagtgt ctttgagtgt tgaagctgct aagactttgg ccgcaaagaa 6420catcaaggct cgtgttgttt ctctaccaga tttcttcact tttgacaaac aacccctaga 6480atacagacta tcagtcttac cagacaacgt tccaatcatg tctgttgaag ttttggctac 6540cacatgttgg ggcaaatacg ctcatcaatc cttcggtatt gacagatttg gtgcctccgg 6600taaggcacca gaagtcttca agttcttcgg tttcacccca gaaggtgttg ctgaaagagc 6660tcaaaagacc attgcattct ataagggtga caagctaatt tctcctttga aaaaagcttt 6720ctaaattctg atcgtagatc atcagatttg atatgatatt atttgtgaaa aaatgaaata 6780aaactttata caacttaaat acaacttttt ttataaacga ttaagcaaaa aaatagtttc 6840aaacttttaa caatattcca aacactcagt ccttttcctt cttatattat aggtgtacgt 6900attatagaaa aatttcaatg attacttttt ctttcttttt ccttgtacca gcacatggcc 6960gagcttgaat gttaaaccct tcgagagaat cacaccattc aagtataaag ccaataaaga 7020atataactcc taaaaggcta attgaaaccc tgtgattttt gcccgggttt aaggcgcgcc 7080ctttatcatt atcaatactg ccatttcaaa gaatacgtaa ataattaata gtagtgattt 7140tcctaacttt atttagtcaa aaaattagcc ttttaattct gctgtaaccc gtacatgccc 7200aaaatagggg gcgggttaca cagaatatat aacatcgtag gtgtctgggt gaacagttta 7260ttcctggcat ccactaaata taatggagcc cgctttttaa gctggcatcc agaaaaaaaa 7320agaatcccag caccaaaata ttgttttctt caccaaccat cagttcatag gtccattctc 7380ttagcgcaac tacagagaac aggggcacaa acaggcaaaa aacgggcaca acctcaatgg 7440agtgatgcaa cctgcctgga gtaaatgatg acacaaggca attgacccac gcatgtatct 7500atctcatttt cttacacctt ctattacctt ctgctctctc tgatttggaa aaagctgaaa 7560aaaaaggttg aaaccagttc cctgaaatta ttcccctact tgactaataa gtatataaag 7620acggtaggta ttgattgtaa ttctgtaaat ctatttctta aacttcttaa attctacttt 7680tatagttagt ctttttttta gttttaaaac accaagaact tagtttcgaa taaacacaca 7740taaacaaaca ccactagcat ggctgccggt gtcccaaaaa ttgatgcgtt agaatctttg 7800ggcaatcctt tggaggatgc caagagagct gcagcataca gagcagttga tgaaaattta 7860aaatttgatg atcacaaaat tattggaatt ggtagtggta gcacagtggt ttatgttgcc 7920gaaagaattg gacaatattt gcatgaccct aaattttatg aagtagcgtc taaattcatt 7980tgcattccaa caggattcca atcaagaaac ttgattttgg ataacaagtt gcaattaggc 8040tccattgaac agtatcctcg cattgatata gcgtttgacg gtgctgatga agtggatgag 8100aatttacaat taattaaagg tggtggtgct tgtctatttc aagaaaaatt ggttagtact 8160agtgctaaaa ccttcattgt cgttgctgat tcaagaaaaa agtcaccaaa acatttaggt 8220aagaactgga ggcaaggtgt tcccattgaa attgtacctt cctcatacgt gagggtcaag 8280aatgatctat tagaacaatt gcatgctgaa aaagttgaca tcagacaagg aggttctgct 8340aaagcaggtc ctgttgtaac tgacaataat aacttcatta tcgatgcgga tttcggtgaa 8400atttccgatc caagaaaatt gcatagagaa atcaaactgt tagtgggcgt ggtggaaaca 8460ggtttattca tcgacaacgc ttcaaaagcc tacttcggta attctgacgg tagtgttgaa 8520gttaccgaaa agtgagcggc cgcgtgaatt tactttaaat cttgcattta aataaatttt 8580ctttttatag ctttatgact tagtttcaat ttatatacta ttttaatgac attttcgatt 8640cattgattga aagctttgtg ttttttcttg atgcgctatt gcattgttct tgtctttttc 8700gccacatgta atatctgtag tagatacctg atacattgtg gatgctgagt gaaattttag 8760ttaataatgg aggcgctctt aataattttg gggatattgg cttttttttt taaagtttac 8820aaatgaattt tttccgccag gataacgatt ctgaagttac tcttagcgtt cctatcggta 8880cagccatcaa atcatgccta taaatcatgc ctatatttgc gtgcagtcag

tatcatctac 8940atgaaaaaaa ctcccgcaat ttcttataga atacgttgaa aattaaatgt acgcgccaag 9000ataagataac atatatctag atgcagtaat atacacagat tcccgcggac gtgggaagga 9060aaaaattaga taacaaaatc tgagtgatat ggaaattccg ctgtatagct catatctttc 9120cctccaccgc ggtggtcgac tttcacatac gttgcatacg tcgatataga taataatgat 9180aatgacagca ggattatcgt aatacgtaat agctgaaaat ctcaaaaatg tgtgggtcat 9240tacgtaaata atgataggaa tgggattctt ctatttttcc tttttccatt ctagcagccg 9300tcgggaaaac gtggcatcct ctctttcggg ctcaattgga gtcacgctgc cgtgagcatc 9360ctctctttcc atatctaaca actgagcacg taaccaatgg aaaagcatga gcttagcgtt 9420gctccaaaaa agtattggat ggttaatacc atttgtctgt tctcttctga ctttgactcc 9480tcaaaaaaaa aaatctacaa tcaacagatc gcttcaatta cgccctcaca aaaacttttt 9540tccttcttct tcgcccacgt taaattttat ccctcatgtt gtctaacgga tttctgcact 9600tgatttatta taaaaagaca aagacataat acttctctat caatttcagt tattgttctt 9660ccttgcgtta ttcttctgtt cttctttttc ttttgtcata tataaccata accaagtaat 9720acatattcaa acttaagact cgagatggtc aaaccaatta tagctcccag tatccttgct 9780tctgacttcg ccaacttggg ttgcgaatgt cataaggtca tcaacgccgg cgcagattgg 9840ttacatatcg atgtcatgga cggccatttt gttccaaaca ttactctggg ccaaccaatt 9900gttacctccc tacgtcgttc tgtgccacgc cctggcgatg ctagcaacac agaaaagaag 9960cccactgcgt tcttcgattg tcacatgatg gttgaaaatc ctgaaaaatg ggtcgacgat 10020tttgctaaat gtggtgctga ccaatttacg ttccactacg aggccacaca agaccctttg 10080catttagtta agttgattaa gtctaagggc atcaaagctg catgcgccat caaacctggt 10140acttctgttg acgttttatt tgaactagct cctcatttgg atatggctct tgttatgact 10200gtggaacctg ggtttggagg ccaaaaattc atggaagaca tgatgccaaa agtggaaact 10260ttgagagcca agttccccca tttgaatatc caagtcgatg gtggtttggg caaggagacc 10320atcccgaaag ccgccaaagc cggtgccaac gttattgtcg ctggtaccag tgttttcact 10380gcagctgacc cgcacgatgt tatctccttc atgaaagaag aagtctcgaa ggaattgcgt 10440tctagagatt tgctagatta gacgtctgtt taaagattac ggatatttaa cttacttaga 10500ataatgccat ttttttgagt tataataatc ctacgttagt gtgagcggga tttaaactgt 10560gaggacctta atacattcag acacttctgc ggtatcaccc tacttattcc cttcgagatt 10620atatctagga acccatcagg ttggtggaag attacccgtt ctaagacttt tcagcttcct 10680ctattgatgt tacacctgga cacccctttt ctggcatcca gtttttaatc ttcagtggca 10740tgtgagattc tccgaaatta attaaagcaa tcacacaatt ctctcggata ccacctcggt 10800tgaaactgac aggtggtttg ttacgcatgc taatgcaaag gagcctatat acctttggct 10860cggctgctgt aacagggaat ataaagggca gcataattta ggagtttagt gaacttgcaa 10920catttactat tttcccttct tacgtaaata tttttctttt taattctaaa tcaatctttt 10980tcaatttttt gtttgtattc ttttcttgct taaatctata actacaaaaa acacatacat 11040aaactaaaac gtacgactag tatgtctgaa ccagctcaaa agaaacaaaa ggttgctaac 11100aactctctag aacaattgaa agcctccggc actgtcgttg ttgccgacac tggtgatttc 11160ggctctattg ccaagtttca acctcaagac tccacaacta acccatcatt gatcttggct 11220gctgccaagc aaccaactta cgccaagttg atcgatgttg ccgtggaata cggtaagaag 11280catggtaaga ccaccgaaga acaagtcgaa aatgctgtgg acagattgtt agtcgaattc 11340ggtaaggaga tcttaaagat tgttccaggc agagtctcca ccgaagttga tgctagattg 11400tcttttgaca ctcaagctac cattgaaaag gctagacata tcattaaatt gtttgaacaa 11460gaaggtgtct ccaaggaaag agtccttatt aaaattgctt ccacttggga aggtattcaa 11520gctgccaaag aattggaaga aaaggacggt atccactgta atttgactct attattctcc 11580ttcgttcaag cagttgcctg tgccgaggcc caagttactt tgatttcccc atttgttggt 11640agaattctag actggtacaa atccagcact ggtaaagatt acaagggtga agccgaccca 11700ggtgttattt ccgtcaagaa aatctacaac tactacaaga agtacggtta caagactatt 11760gttatgggtg cttctttcag aagcactgac gaaatcaaaa acttggctgg tgttgactat 11820ctaacaattt ctccagcttt attggacaag ttgatgaaca gtactgaacc tttcccaaga 11880gttttggacc ctgtctccgc taagaaggaa gccggcgaca agatttctta catcagcgac 11940gaatctaaat tcagattcga cttgaatgaa gacgctatgg ccactgaaaa attgtccgaa 12000ggtatcagaa aattctctgc cgatattgtt actctattcg acttgattga aaagaaagtt 12060accgcttaag gaagtatctc ggaaatatta atttaggcca tgtccttatg cacgtttctt 12120ttgatactta cgggtacatg tacacaagta tatctatata tataaattaa tgaaaatccc 12180ctatttatat atatgacttt aacgagacag aacagttttt tattttttat cctatttgat 12240gaatgataca gtttcggatc cacgatcgca ttgcggatta cgtattctaa tgttcagtac 12300cgttcgtata atgtatgcta tacgaagtta tgcagattgt actgagagtg caccatacca 12360cagcttttca attcaattca tcattttttt tttattcttt tttttgattt cggtttcttt 12420gaaatttttt tgattcggta atctccgaac agaaggaaga acgaaggaag gagcacagac 12480ttagattggt atatatacgc atatgtagtg ttgaagaaac atgaaattgc ccagtattct 12540taacccaact gcacagaaca aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta 12600catataagga acgtgctgct actcatccta gtcctgttgc tgccaagcta tttaatatca 12660tgcacgaaaa gcaaacaaac ttgtgtgctt cattggatgt tcgtaccacc aaggaattac 12720tggagttagt tgaagcatta ggtcccaaaa tttgtttact aaaaacacat gtggatatct 12780tgactgattt ttccatggag ggcacagtta agccgctaaa ggcattatcc gccaagtaca 12840attttttact cttcgaagac agaaaatttg ctgacattgg taatacagtc aaattgcagt 12900actctgcggg tgtatacaga atagcagaat gggcagacat tacgaatgca cacggtgtgg 12960tgggcccagg tattgttagc ggtttgaagc aggcggcaga agaagtaaca aaggaaccta 13020gaggcctttt gatgttagca gaattgtcat gcaagggctc cctatctact ggagaatata 13080ctaagggtac tgttgacatt gcgaagagcg acaaagattt tgttatcggc tttattgctc 13140aaagagacat gggtggaaga gatgaaggtt acgattggtt gattatgaca cccggtgtgg 13200gtttagatga caagggagac gcattgggtc aacagtatag aaccgtggat gatgtggtct 13260ctacaggatc tgacattatt attgttggaa gaggactatt tgcaaaggga agggatgcta 13320aggtagaggg tgaacgttac agaaaagcag gctgggaagc atatttgaga agatgcggcc 13380agcaaaacta aaaaactgta ttataagtaa atgcatgtat actaaactca caaattagag 13440cttcaattta attatatcag ttattaccct atgcggtgtg aaataccgca cagatgcgta 13500aggagaaaat accgcatcag gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa 13560atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 13620aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 13680tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 13740cactacgtga accatcaccc taatcaagat aacttcgtat aatgtatgct atacgaacgg 13800tacccgccaa ctctgttcga gaatgatgta atcaagaagg tctcacaaaa ccatccaggc 13860agtaccactt cccaagtatt gcttagatgg gcaactcaga gaggcattgc cgtcattcca 13920aaatcttcca agaaggaaag gttacttggc aacctagaaa tcgaaaaaaa gttcacttta 13980acggagcaag aattgaagga tatttctgca ctaaatgcca acatcagatt taatgatcca 14040tggacctggt tggatggtaa attccccact tttgcctgat ccagccagta aaatccatac 14100tcaacgacga tatgaacaaa tttccctcat tccgatgctg tatatgtgta taaattttta 14160catgctcttc tgtttagaca cagaacagct ttaaataaaa tgttggatat actttttctg 14220cctgtggtgt catccacgct tttaattcat ctcttgtatg gttgacaatt tggctatttt 14280ttaacagaac ccaacggtaa ttgaaattaa aagggaaacg agtgggggcg atgagtgagt 14340gatacggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 14400atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc 14460gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 14520agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 14580cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 14640ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 14700atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 14760tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 14820cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 14880agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 14940taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 15000tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 15060catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 15120ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 15180ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 15240catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 15300aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 15360aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 15420taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 15480atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 15540gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 15600tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 15660ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 15720gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 15780agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 15840aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 15900agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 15960tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 16020atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 16080taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 16140gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 16200gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 16260aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 16320tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 16380gtcagggggg cggagcctat ggaa 1640444440PRTartificial sequenceconstructed xylose isomerase 44Met Ala Lys Glu Tyr Phe Pro Gln Ile Gln Lys Ile Gln Tyr Gln Gly1 5 10 15Pro Lys Ser Thr Asp Pro Leu Ser Phe Lys Tyr Tyr Asn Pro Glu Glu 20 25 30Val Ile Asn Gly Lys Thr Met Arg Glu His Leu Lys Phe Ala Leu Ser 35 40 45Trp Trp His Thr Met Gly Gly Asp Gly Thr Asp Met Phe Gly Cys Gly 50 55 60Thr Thr Asp Lys Thr Trp Gly Gln Ser Asp Pro Ala Ala Arg Ala Lys65 70 75 80Ala Lys Val Asp Ala Ala Phe Glu Ile Met Asp Lys Leu Ser Ile Asp 85 90 95Tyr Tyr Cys Phe His Asp Arg Asp Leu Ser Pro Glu Tyr Gly Ser Leu 100 105 110Lys Ala Thr Asn Asp Gln Leu Asp Ile Val Thr Asp Tyr Ile Lys Glu 115 120 125Lys Gln Gly Asp Lys Phe Lys Cys Leu Trp Gly Thr Ala Lys Cys Phe 130 135 140Asp His Pro Arg Phe Met His Gly Ala Gly Thr Ser Pro Ser Ala Asp145 150 155 160Val Phe Ala Phe Ser Ala Ala Gln Ile Lys Lys Ala Leu Glu Ser Thr 165 170 175Val Lys Leu Gly Ala Asn Gly Tyr Val Phe Trp Gly Gly Arg Glu Gly 180 185 190Tyr Glu Thr Leu Leu Asn Thr Asn Met Gly Leu Glu Leu Asp Asn Met 195 200 205Ala Arg Leu Met Lys Met Ala Val Glu Tyr Gly Arg Ser Ile Gly Phe 210 215 220Lys Gly Asp Phe Tyr Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys His225 230 235 240Gln Tyr Asp Phe Asp Thr Ala Thr Val Leu Gly Phe Leu Arg Lys Tyr 245 250 255Gly Leu Asp Lys Asp Phe Lys Met Asn Ile Glu Ala Asn His Ala Thr 260 265 270Leu Ala Gln His Thr Phe Gln His Glu Leu Arg Val Ala Arg Asp Asn 275 280 285Gly Val Phe Gly Ser Ile Asp Ala Asn Gln Gly Asp Val Leu Leu Gly 290 295 300Trp Asp Thr Asp Gln Phe Pro Thr Asn Ile Tyr Asp Thr Thr Met Cys305 310 315 320Met Tyr Glu Val Ile Lys Ala Gly Gly Phe Thr Asn Gly Gly Leu Asn 325 330 335Phe Asp Ala Lys Ala Arg Arg Gly Ser Phe Thr Pro Glu Asp Ile Phe 340 345 350Tyr Ser Tyr Ile Ala Gly Met Asp Ala Phe Ala Leu Gly Phe Arg Ala 355 360 365Ala Leu Lys Leu Ile Glu Asp Gly Arg Ile Asp Lys Phe Val Ala Asp 370 375 380Arg Tyr Ala Ser Trp Asn Thr Gly Ile Gly Ala Asp Ile Ile Ala Gly385 390 395 400Lys Ala Asp Phe Ala Ser Leu Glu Lys Tyr Ala Leu Glu Lys Gly Glu 405 410 415Val Thr Ala Ser Leu Ser Ser Gly Arg Gln Glu Met Leu Glu Ser Ile 420 425 430Val Asn Asn Val Leu Phe Ser Leu 435 440451323DNAartificial sequencecodon optimized coding region for constructed xylose isomerase VDxylA 45atggctaagg aatacttccc acaaatccaa aagatccaat accaaggtcc aaagtccact 60gacccattgt ccttcaagta ctacaaccca gaagaagtca tcaacggtaa aaccatgaga 120gaacacttga agttcgcttt gtcttggtgg cacaccatgg gtggtgacgg tactgatatg 180ttcggttgtg gtactactga caagacttgg ggtcaatctg atccagctgc tagagctaag 240gctaaggttg acgctgcttt cgaaatcatg gacaagttgt ccatcgatta ctactgtttc 300cacgacagag atttgtctcc agaatacggt tccttgaagg ctaccaacga ccaattggat 360atcgtcactg actacatcaa ggaaaagcaa ggtgacaagt tcaagtgttt gtggggtact 420gctaagtgtt tcgaccaccc aagattcatg cacggtgctg gtacttctcc atccgctgac 480gttttcgctt tctctgctgc tcaaatcaag aaggctttgg aatccaccgt taagttgggt 540gctaacggtt acgtcttctg gggtggtaga gaaggttacg aaaccttgtt gaacactaac 600atgggtttgg aattggacaa catggctaga ttgatgaaga tggctgttga atacggtaga 660tctatcggtt tcaagggtga cttctacatc gaaccaaagc caaaggaacc aactaagcac 720caatacgact tcgataccgc tactgtcttg ggtttcttga gaaagtacgg tttggacaag 780gatttcaaga tgaacatcga agctaaccac gctaccttgg ctcaacacac tttccaacac 840gaattgagag ttgctagaga caacggtgtc ttcggttcca tcgatgctaa ccaaggtgac 900gttttgttgg gttgggacac cgatcaattc ccaactaaca tctacgacac cactatgtgt 960atgtacgaag tcatcaaggc tggtggtttc accaacggtg gtttgaactt cgacgctaag 1020gctagaagag gttctttcac tccagaagac atcttctact cctacatcgc tggtatggac 1080gctttcgctt tgggtttcag agctgctttg aagttgatcg aagacggtag aatcgataag 1140ttcgttgctg acagatacgc ttcttggaac accggtatcg gtgctgacat catcgctggt 1200aaagctgatt tcgcttcttt ggaaaagtac gctttggaaa agggtgaagt cactgcttcc 1260ttgtcctctg gtagacaaga aatgttggaa tccatcgtca acaacgtttt gttctctttg 1320tga 1323462966DNAartificial sequenceconstructed chimeric expression cassette for VDxykA 46tgacagcagg attatcgtaa tacgtaatag ttgaaaatct caaaaatgtg tgggtcatta 60cgtaaataat gataggaatg ggattcttct atttttcctt tttccattct gtcgaccgca 120cgccgaaatg catgcaagta acctattcaa agtaatatct catacatgtt tcatgagggt 180aacaacatgc gactgggtga gcatatgttc cgctgatgtg atgtgcaaga taaacaagca 240agacagaaac taacttcttc ttcatgtaat aaacacaccc cgcgtttatt tacctatctt 300taaacttcaa caccttatat cataactaat atttcttgag ataagcacac tgcacccata 360ccttccttaa aaacgtagct tccagttttt ggtggttctg gcttccttcc cgattccgcc 420cgctaaacgc ataattttgt tgcctggtgg catttgcaaa atgcataacc tatgcattta 480aaagattatg tatgctcttc tgacttttcg tgtgatgagg ctcgtggaaa aaatgaataa 540tttatgaatt tgagaacaat tttgtgttgt tacggtattt tactatggaa taatcaatca 600attgaggatt ttatgcaaat atcgtttgaa tatttttccg accctttgag tacttttctt 660cataattgca taatattgtc cgctgcccgt ttttctgtta gacggtgtct tgatctactt 720gctatcgttc aacaccacct tatcttctaa ctattttttt tttagctcat ttgaatcagc 780ttatggtgat ggcacatttt tgcataaacc tagctgtcct cgttgaacat aggaaaaaaa 840aatatataaa caaggctctt tcactctcct tggaatcaga tttgggtttg ttccctttat 900tttcatattt cttgtcatat tcttttctca attattattt tctactcata acctcacgca 960aaataacaca gtcaaatcaa tcaagtttaa acagtatggc taaggaatac ttcccacaaa 1020tccaaaagat ccaataccaa ggtccaaagt ccactgaccc attgtccttc aagtactaca 1080acccagaaga agtcatcaac ggtaaaacca tgagagaaca cttgaagttc gctttgtctt 1140ggtggcacac catgggtggt gacggtactg atatgttcgg ttgtggtact actgacaaga 1200cttggggtca atctgatcca gctgctagag ctaaggctaa ggttgacgct gctttcgaaa 1260tcatggacaa gttgtccatc gattactact gtttccacga cagagatttg tctccagaat 1320acggttcctt gaaggctacc aacgaccaat tggatatcgt cactgactac atcaaggaaa 1380agcaaggtga caagttcaag tgtttgtggg gtactgctaa gtgtttcgac cacccaagat 1440tcatgcacgg tgctggtact tctccatccg ctgacgtttt cgctttctct gctgctcaaa 1500tcaagaaggc tttggaatcc accgttaagt tgggtgctaa cggttacgtc ttctggggtg 1560gtagagaagg ttacgaaacc ttgttgaaca ctaacatggg tttggaattg gacaacatgg 1620ctagattgat gaagatggct gttgaatacg gtagatctat cggtttcaag ggtgacttct 1680acatcgaacc aaagccaaag gaaccaacta agcaccaata cgacttcgat accgctactg 1740tcttgggttt cttgagaaag tacggtttgg acaaggattt caagatgaac atcgaagcta 1800accacgctac cttggctcaa cacactttcc aacacgaatt gagagttgct agagacaacg 1860gtgtcttcgg ttccatcgat gctaaccaag gtgacgtttt gttgggttgg gacaccgatc 1920aattcccaac taacatctac gacaccacta tgtgtatgta cgaagtcatc aaggctggtg 1980gtttcaccaa cggtggtttg aacttcgacg ctaaggctag aagaggttct ttcactccag 2040aagacatctt ctactcctac atcgctggta tggacgcttt cgctttgggt ttcagagctg 2100ctttgaagtt gatcgaagac ggtagaatcg ataagttcgt tgctgacaga tacgcttctt 2160ggaacaccgg tatcggtgct gacatcatcg ctggtaaagc tgatttcgct tctttggaaa 2220agtacgcttt ggaaaagggt gaagtcactg cttccttgtc ctctggtaga caagaaatgt 2280tggaatccat cgtcaacaac gttttgttct ctttgtgagg ccctgcaggc cagaggaaaa 2340taatatcaag tgctggaaac tttttctctt ggaatttttg caacatcaag tcatagtcaa 2400ttgaattgac ccaatttcac atttaagatt tttttttttt catccgacat acatctgtac 2460actaggaagc cctgtttttc tgaagcagct tcaaatatat atatttttta catatttatt 2520atgattcaat gaacaatcta attaaatcga aaacaagaac cgaaacgcga ataaataatt 2580tatttagatg gtgacaagtg tataagtcct catcgggaca gctacgattt ctctttcggt 2640tttggctgag ctactggttg ctgtgacgca gcggcattag cgcggcgtta tgagctaccc 2700tcgtggcctg aaagatggcg ggaataaagc ggaactaaaa attactgact gagccatatt 2760gaggtcaatt tgtcaactcg tcaagtcacg tttggtggac ggcccctttc caacgaatcg 2820tatatactaa catgcgcgcg cttcctatat acacatatac atatatatat atatatatat 2880gtgtgcgtgt atgtgtacac ctgtatttaa tttccttact cgcgggtttt tcttttttct 2940caattcttgg cttcctcttt ctcgag 2966478601DNAartificial sequenceconstructed plasmid 47aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 60gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 120tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 180agagcgccca atacgcaaac cgcctctccc cgcgcgttgg

ccgattcatt aatgcagctg 240gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 300gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 360aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 420tggcgccact tgtgcatgat taccgacaac caaaaccagt cacagattct attaatcctc 480caaatgtaaa cataaccacc tccacaacca acaagaacct agatggcatt tatattttgc 540cagctcctcg tatgaatccc ccggctcaaa cacaatacca aatgattcat gcgccagaca 600gcatgcaaca tccaccaaca tttagtaaaa acaacacatc aagcaatcct aaatcccacc 660aatactcaaa gtagaagatc agcatccttt caattgctga aaggttcacc taaagtaccg 720ctcatattcc aaaaggattc ttcactacat agaaagggca gccaattgtg tgtttttcag 780aaagggtttt aaaaaaacag gagggtgctt gttcttgttg ttccctacca tcgatggatt 840tcgaaaaact atttatagga ccatctgatt ttcacctcca tcattgtatc atatactaac 900aagcatatcc aaatttgtaa ttctatcatg aaatttccag agaaagaaac gcaagggaac 960tgagaaatca aacactagtt gacagcagga ttatcgtaat acgtaatagt tgaaaatctc 1020aaaaatgtgt gggtcattac gtaaataatg ataggaatgg gattcttcta tttttccttt 1080ttccattctg tcgaccgcac gccgaaatgc atgcaagtaa cctattcaaa gtaatatctc 1140atacatgttt catgagggta acaacatgcg actgggtgag catatgttcc gctgatgtga 1200tgtgcaagat aaacaagcaa gacagaaact aacttcttct tcatgtaata aacacacccc 1260gcgtttattt acctatcttt aaacttcaac accttatatc ataactaata tttcttgaga 1320taagcacact gcacccatac cttccttaaa aacgtagctt ccagtttttg gtggttctgg 1380cttccttccc gattccgccc gctaaacgca taattttgtt gcctggtggc atttgcaaaa 1440tgcataacct atgcatttaa aagattatgt atgctcttct gacttttcgt gtgatgaggc 1500tcgtggaaaa aatgaataat ttatgaattt gagaacaatt ttgtgttgtt acggtatttt 1560actatggaat aatcaatcaa ttgaggattt tatgcaaata tcgtttgaat atttttccga 1620ccctttgagt acttttcttc ataattgcat aatattgtcc gctgcccgtt tttctgttag 1680acggtgtctt gatctacttg ctatcgttca acaccacctt atcttctaac tatttttttt 1740ttagctcatt tgaatcagct tatggtgatg gcacattttt gcataaacct agctgtcctc 1800gttgaacata ggaaaaaaaa atatataaac aaggctcttt cactctcctt ggaatcagat 1860ttgggtttgt tccctttatt ttcatatttc ttgtcatatt cttttctcaa ttattatttt 1920ctactcataa cctcacgcaa aataacacag tcaaatcaat caagtttaaa cagtatggct 1980aaggaatact tcccacaaat ccaaaagatc caataccaag gtccaaagtc cactgaccca 2040ttgtccttca agtactacaa cccagaagaa gtcatcaacg gtaaaaccat gagagaacac 2100ttgaagttcg ctttgtcttg gtggcacacc atgggtggtg acggtactga tatgttcggt 2160tgtggtacta ctgacaagac ttggggtcaa tctgatccag ctgctagagc taaggctaag 2220gttgacgctg ctttcgaaat catggacaag ttgtccatcg attactactg tttccacgac 2280agagatttgt ctccagaata cggttccttg aaggctacca acgaccaatt ggatatcgtc 2340actgactaca tcaaggaaaa gcaaggtgac aagttcaagt gtttgtgggg tactgctaag 2400tgtttcgacc acccaagatt catgcacggt gctggtactt ctccatccgc tgacgttttc 2460gctttctctg ctgctcaaat caagaaggct ttggaatcca ccgttaagtt gggtgctaac 2520ggttacgtct tctggggtgg tagagaaggt tacgaaacct tgttgaacac taacatgggt 2580ttggaattgg acaacatggc tagattgatg aagatggctg ttgaatacgg tagatctatc 2640ggtttcaagg gtgacttcta catcgaacca aagccaaagg aaccaactaa gcaccaatac 2700gacttcgata ccgctactgt cttgggtttc ttgagaaagt acggtttgga caaggatttc 2760aagatgaaca tcgaagctaa ccacgctacc ttggctcaac acactttcca acacgaattg 2820agagttgcta gagacaacgg tgtcttcggt tccatcgatg ctaaccaagg tgacgttttg 2880ttgggttggg acaccgatca attcccaact aacatctacg acaccactat gtgtatgtac 2940gaagtcatca aggctggtgg tttcaccaac ggtggtttga acttcgacgc taaggctaga 3000agaggttctt tcactccaga agacatcttc tactcctaca tcgctggtat ggacgctttc 3060gctttgggtt tcagagctgc tttgaagttg atcgaagacg gtagaatcga taagttcgtt 3120gctgacagat acgcttcttg gaacaccggt atcggtgctg acatcatcgc tggtaaagct 3180gatttcgctt ctttggaaaa gtacgctttg gaaaagggtg aagtcactgc ttccttgtcc 3240tctggtagac aagaaatgtt ggaatccatc gtcaacaacg ttttgttctc tttgtgaggc 3300cctgcaggcc agaggaaaat aatatcaagt gctggaaact ttttctcttg gaatttttgc 3360aacatcaagt catagtcaat tgaattgacc caatttcaca tttaagattt tttttttttc 3420atccgacata catctgtaca ctaggaagcc ctgtttttct gaagcagctt caaatatata 3480tattttttac atatttatta tgattcaatg aacaatctaa ttaaatcgaa aacaagaacc 3540gaaacgcgaa taaataattt atttagatgg tgacaagtgt ataagtcctc atcgggacag 3600ctacgatttc tctttcggtt ttggctgagc tactggttgc tgtgacgcag cggcattagc 3660gcggcgttat gagctaccct cgtggcctga aagatggcgg gaataaagcg gaactaaaaa 3720ttactgactg agccatattg aggtcaattt gtcaactcgt caagtcacgt ttggtggacg 3780gcccctttcc aacgaatcgt atatactaac atgcgcgcgc ttcctatata cacatataca 3840tatatatata tatatatatg tgtgcgtgta tgtgtacacc tgtatttaat ttccttactc 3900gcgggttttt cttttttctc aattcttggc ttcctctttc tcgagcccgg gatttaaatg 3960tggcttactc cattgttgat gcaaaagttg taaatttcac gaattattta atgcgttcct 4020tgcaaccttc tattttgatg aacatcagga attgaaacaa aaaaaaggct tcaatctcaa 4080cggaaaacgg gaagaaaact acactcgatt atactatata tgccaagaag attctccgac 4140agattgtcta cttaatttca cataatatat cttgttttac tagcttatta tatagcgtcg 4200catttaattc atggcgccat cacccgcagg gaatataacg acaaggccga taccacggga 4260aaaatagggc gagcggaaat actaaaagaa aaataagctt ccgaaataaa acaccgacaa 4320tgaagttctt ggcaaggttc ggttaggatc cgcattgcgg attacgtatt ctaatgttca 4380gtaccgttcg tataatgtat gctatacgaa gttatgcaga ttgtactgag agtgcaccat 4440accacagctt ttcaattcaa ttcatcattt tttttttatt cttttttttg atttcggttt 4500ctttgaaatt tttttgattc ggtaatctcc gaacagaagg aagaacgaag gaaggagcac 4560agacttagat tggtatatat acgcatatgt agtgttgaag aaacatgaaa ttgcccagta 4620ttcttaaccc aactgcacag aacaaaaacc tgcaggaaac gaagataaat catgtcgaaa 4680gctacatata aggaacgtgc tgctactcat cctagtcctg ttgctgccaa gctatttaat 4740atcatgcacg aaaagcaaac aaacttgtgt gcttcattgg atgttcgtac caccaaggaa 4800ttactggagt tagttgaagc attaggtccc aaaatttgtt tactaaaaac acatgtggat 4860atcttgactg atttttccat ggagggcaca gttaagccgc taaaggcatt atccgccaag 4920tacaattttt tactcttcga agacagaaaa tttgctgaca ttggtaatac agtcaaattg 4980cagtactctg cgggtgtata cagaatagca gaatgggcag acattacgaa tgcacacggt 5040gtggtgggcc caggtattgt tagcggtttg aagcaggcgg cagaagaagt aacaaaggaa 5100cctagaggcc ttttgatgtt agcagaattg tcatgcaagg gctccctatc tactggagaa 5160tatactaagg gtactgttga cattgcgaag agcgacaaag attttgttat cggctttatt 5220gctcaaagag acatgggtgg aagagatgaa ggttacgatt ggttgattat gacacccggt 5280gtgggtttag atgacaaggg agacgcattg ggtcaacagt atagaaccgt ggatgatgtg 5340gtctctacag gatctgacat tattattgtt ggaagaggac tatttgcaaa gggaagggat 5400gctaaggtag agggtgaacg ttacagaaaa gcaggctggg aagcatattt gagaagatgc 5460ggccagcaaa actaaaaaac tgtattataa gtaaatgcat gtatactaaa ctcacaaatt 5520agagcttcaa tttaattata tcagttatta ccctatgcgg tgtgaaatac cgcacagatg 5580cgtaaggaga aaataccgca tcaggaaatt gtaaacgtta atattttgtt aaaattcgcg 5640ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct 5700tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt 5760ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat 5820ggcccactac gtgaaccatc accctaatca agataacttc gtataatgta tgctatacga 5880acggtaccga gatacccact tcgaaagtta ctgatattat aactcttgtg tcctctcttc 5940taatacctta ctttcacctt tctcacgtag ttaaagttgc aacaacacat tttgtcctca 6000tccaatttct tctatagaat atccgtttgc ctccaggagt gaagaaatga tagcagtaac 6060actgtgaaag cgagactaag agaaacgact taaagctcga agacttcttg aggatacgtt 6120tatgtttctg tggcttcttc ttcgcggcgc ggttctcgcg tataggaatg ttctaagaca 6180agaaggcatg aagttatgtt aacagattct atatctactc gctacgcata tataaacgga 6240ttcatcattg aaacaatggt acttgtggta atgtgtacga cgatttcaac ccgaataaaa 6300gcaaaagtgc aaaaaaaaaa caagaagcgc ttagcactgt tgaatcattt agaacactac 6360taatgctggt aatacggcgc cgaattcact ggccgtcgtt ttacaacgtc gtgactggga 6420aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg 6480taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 6540atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 6600gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 6660aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 6720tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 6780gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 6840ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 6900tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 6960ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 7020ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 7080tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 7140gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 7200gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 7260acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 7320tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 7380caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 7440gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 7500cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 7560tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 7620agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 7680tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 7740ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 7800acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 7860ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 7920gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 7980gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 8040ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 8100gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 8160ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 8220cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 8280cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 8340ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 8400tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 8460cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 8520ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 8580aggggggcgg agcctatgga a 8601488645DNAartificial sequenceconstructed plasmid 48accttggctc aacacacttt ccaacacgaa ttgagagttg ctagagacaa cggtgtcttc 60ggttccatcg atgctaacca aggtgacgtt ttgttgggtt gggacaccga tcaattccca 120actaacatct acgacaccac tatgtgtatg tacgaagtca tcaaggctgg tggtttcacc 180aacggtggtt tgaacttcga cgctaaggct agaagaggtt ctttcactcc agaagacatc 240ttctactcct acatcgctgg tatggacgct ttcgctttgg gtttcagagc tgctttgaag 300ttgatcgaag acggtagaat cgataagttc gttgctgaca gatacgcttc ttggaacacc 360ggtatcggtg ctgacatcat cgctggtaaa gctgatttcg cttctttgga aaagtacgct 420ttggaaaagg gtgaagtcac tgcttccttg tcctctggta gacaagaaat gttggaatcc 480atcgtcaaca acgttttgtt ctctttgtga ggccctgcag gccagaggaa aataatatca 540agtgctggaa actttttctc ttggaatttt tgcaacatca agtcatagtc aattgaattg 600acccaatttc acatttaaga tttttttttt ttcatccgac atacatctgt acactaggaa 660gccctgtttt tctgaagcag cttcaaatat atatattttt tacatattta ttatgattca 720atgaacaatc taattaaatc gaaaacaaga accgaaacgc gaataaataa tttatttaga 780tggtgacaag tgtataagtc ctcatcggga cagctacgat ttctctttcg gttttggctg 840agctactggt tgctgtgacg cagcggcatt agcgcggcgt tatgagctac cctcgtggcc 900tgaaagatgg cgggaataaa gcggaactaa aaattactga ctgagccata ttgaggtcaa 960tttgtcaact cgtcaagtca cgtttggtgg acggcccctt tccaacgaat cgtatatact 1020aacatgcgcg cgcttcctat atacacatat acatatatat atatatatat atgtgtgcgt 1080gtatgtgtac acctgtattt aatttcctta ctcgcgggtt tttctttttt ctcaattctt 1140ggcttcctct ttctcgagcc cgggatttaa atccttatac cctcatctta ccgcagtgcg 1200gttttacgcc tcatgttatt tttgccagct ttaataacaa catcagtaat attacgttat 1260tggatcttcc actccggttc gaggaaaaaa agagaggagg agaaacgcat aagctacaat 1320aatgagtgag ttaacgcttt aatttgctca gtgatcattg ctagccggat atttgtgttt 1380ttgtagaacc cagccatacc taatcatctc agtatattat ctgaattctt gactcaaaat 1440atggattttc tcgaacgtct cacttccaat catcccatca aatgctgtgg atgaaaacat 1500ttaaaacacg ggcaagaaaa aatgagtata gtatagttgt ggccagttct tgttgtacta 1560atgcacttgc tttcgtatca aagttgaaga agcactgaaa gaacaacaaa aaatttatta 1620aaagcaaaaa tcattctacg ttcaacgaaa atgtaaggca aatatatctt tatataggca 1680agcataaccg acgcggatcc gcattgcgga ttacgtattc taatgttcag taccgttcgt 1740ataatgtatg ctatacgaag ttatgcagat tgtactgaga gtgcaccata ccacagcttt 1800tcaattcaat tcatcatttt ttttttattc ttttttttga tttcggtttc tttgaaattt 1860ttttgattcg gtaatctccg aacagaagga agaacgaagg aaggagcaca gacttagatt 1920ggtatatata cgcatatgta gtgttgaaga aacatgaaat tgcccagtat tcttaaccca 1980actgcacaga acaaaaacct gcaggaaacg aagataaatc atgtcgaaag ctacatataa 2040ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata tcatgcacga 2100aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat tactggagtt 2160agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata tcttgactga 2220tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt acaatttttt 2280actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc agtactctgc 2340gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg tggtgggccc 2400aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac ctagaggcct 2460tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat atactaaggg 2520tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg ctcaaagaga 2580catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg tgggtttaga 2640tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg tctctacagg 2700atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg ctaaggtaga 2760gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg gccagcaaaa 2820ctaaaaaact gtattataag taaatgcatg tatactaaac tcacaaatta gagcttcaat 2880ttaattatat cagttattac cctatgcggt gtgaaatacc gcacagatgc gtaaggagaa 2940aataccgcat caggaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg 3000ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 3060agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 3120gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 3180tgaaccatca ccctaatcaa gataacttcg tataatgtat gctatacgaa cggtacctga 3240aaggtttacg atattagctg ctaagttctc ccttgcccta gtgaattacg tttttttcga 3300ttaaaagggt tggtcccgaa attgacctct tatatgtacc tcaattgcag atagcatcaa 3360atttagacta cgtcatcaac caaattacac cctatgaaaa acataagatt ataagcatgc 3420gtttgtgtaa aggtcatacc attttatacg ttgacgcaat ggtgcaaata gaaaggttta 3480cgaaagctct tattccgctt agcgcaattc tattgttaat ttcaagtaaa aagataaata 3540tctcacgtcc ttatattatc tttgattaaa atttcacgat caatgtaacg aaaaaaggtc 3600gcaaatattt ctttttcatc actctgatta aacaatggca tcatgaaacg ctcattggat 3660cgttacacgt tctcattgta catatgtatg atttagaaca gcttgttcgc ctgggcgccg 3720aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt 3780aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 3840gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt 3900ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 3960tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 4020cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 4080atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 4140cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 4200tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 4260tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 4320atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4380gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4440cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 4500gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 4560cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 4620gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 4680tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 4740ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 4800gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 4860cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 4920tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 4980tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 5040cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 5100acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 5160tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 5220ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5280accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5340aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5400ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5460gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 5520ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5580ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5640ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5700gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 5760cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 5820cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 5880cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 5940aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 6000ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 6060gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 6120gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 6180cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag 6240ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga 6300attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt 6360ggcgcccgag gtttgagtca atgtacgaga ttttgaaacc atgagggatg tgccctcaaa 6420ctttatgatc tggtttcttt ttttatttat atcgttttta tgctgggtta cgttgttgtt 6480ttatattcct tgtctagtat tacggtcagc agaggccaaa tcctatatgc attatatgtg 6540gttagcattg actccttaat agaacggcca gttatcaatc aaattgccta taagcatgga

6600tcactagcgg ctagtattgt attgcatttg gaatagccct cttcgttgag ccacaaagat 6660atcttgcgga aatgtggcta aagcaacttt tgatgattgc tgggaaacgg tttggtaaac 6720tgctgaagca attgttgtac acaatacttc tgcttttggc cccatctgga tttttcattt 6780ccgcttttgc ctcaggtgca acaataacta tctttgagca tcgtaccacc aactagttga 6840cagcaggatt atcgtaatac gtaatagttg aaaatctcaa aaatgtgtgg gtcattacgt 6900aaataatgat aggaatggga ttcttctatt tttccttttt ccattctgtc gaccgcacgc 6960cgaaatgcat gcaagtaacc tattcaaagt aatatctcat acatgtttca tgagggtaac 7020aacatgcgac tgggtgagca tatgttccgc tgatgtgatg tgcaagataa acaagcaaga 7080cagaaactaa cttcttcttc atgtaataaa cacaccccgc gtttatttac ctatctttaa 7140acttcaacac cttatatcat aactaatatt tcttgagata agcacactgc acccatacct 7200tccttaaaaa cgtagcttcc agtttttggt ggttctggct tccttcccga ttccgcccgc 7260taaacgcata attttgttgc ctggtggcat ttgcaaaatg cataacctat gcatttaaaa 7320gattatgtat gctcttctga cttttcgtgt gatgaggctc gtggaaaaaa tgaataattt 7380atgaatttga gaacaatttt gtgttgttac ggtattttac tatggaataa tcaatcaatt 7440gaggatttta tgcaaatatc gtttgaatat ttttccgacc ctttgagtac ttttcttcat 7500aattgcataa tattgtccgc tgcccgtttt tctgttagac ggtgtcttga tctacttgct 7560atcgttcaac accaccttat cttctaacta tttttttttt agctcatttg aatcagctta 7620tggtgatggc acatttttgc ataaacctag ctgtcctcgt tgaacatagg aaaaaaaaat 7680atataaacaa ggctctttca ctctccttgg aatcagattt gggtttgttc cctttatttt 7740catatttctt gtcatattct tttctcaatt attattttct actcataacc tcacgcaaaa 7800taacacagtc aaatcaatca agtttaaaca gtatggctaa ggaatacttc ccacaaatcc 7860aaaagatcca ataccaaggt ccaaagtcca ctgacccatt gtccttcaag tactacaacc 7920cagaagaagt catcaacggt aaaaccatga gagaacactt gaagttcgct ttgtcttggt 7980ggcacaccat gggtggtgac ggtactgata tgttcggttg tggtactact gacaagactt 8040ggggtcaatc tgatccagct gctagagcta aggctaaggt tgacgctgct ttcgaaatca 8100tggacaagtt gtccatcgat tactactgtt tccacgacag agatttgtct ccagaatacg 8160gttccttgaa ggctaccaac gaccaattgg atatcgtcac tgactacatc aaggaaaagc 8220aaggtgacaa gttcaagtgt ttgtggggta ctgctaagtg tttcgaccac ccaagattca 8280tgcacggtgc tggtacttct ccatccgctg acgttttcgc tttctctgct gctcaaatca 8340agaaggcttt ggaatccacc gttaagttgg gtgctaacgg ttacgtcttc tggggtggta 8400gagaaggtta cgaaaccttg ttgaacacta acatgggttt ggaattggac aacatggcta 8460gattgatgaa gatggctgtt gaatacggta gatctatcgg tttcaagggt gacttctaca 8520tcgaaccaaa gccaaaggaa ccaactaagc accaatacga cttcgatacc gctactgtct 8580tgggtttctt gagaaagtac ggtttggaca aggatttcaa gatgaacatc gaagctaacc 8640acgct 8645498537DNAartificial sequenceconstructed plasmid 49aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 60gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc 120tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga 180agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg 240gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta 300gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg 360aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct 420tggcgccttg agcattagtt gatcattacc gctgcagtca gttaatgata tccctaaata 480ggcatattgt acaatttcaa atatattacc cgaattacaa gaacaatagg gtcttctaca 540atattactat gaacaattca taggggaaca tagtccactt caatgtcggt gccaacgatt 600ctatttgttg taagtatata tgctgtttcc cggcaatact ttatattggg ggaattttct 660ggaggtttaa gggcatatta ctacgtcaag gctgggattg accttctggt atatattacg 720ttgcgcagga cattccttat acttgagcct ttgtaaaaac ttcttcgttc tcctgaccaa 780ccgttcaatt atgttgtgaa ttttttgatc cgagtttgaa acatccaaat tcacaaacaa 840accgttgtca tcctgagttg gcgtactagt tgacagcagg attatcgtaa tacgtaatag 900ttgaaaatct caaaaatgtg tgggtcatta cgtaaataat gataggaatg ggattcttct 960atttttcctt tttccattct gtcgaccgca cgccgaaatg catgcaagta acctattcaa 1020agtaatatct catacatgtt tcatgagggt aacaacatgc gactgggtga gcatatgttc 1080cgctgatgtg atgtgcaaga taaacaagca agacagaaac taacttcttc ttcatgtaat 1140aaacacaccc cgcgtttatt tacctatctt taaacttcaa caccttatat cataactaat 1200atttcttgag ataagcacac tgcacccata ccttccttaa aaacgtagct tccagttttt 1260ggtggttctg gcttccttcc cgattccgcc cgctaaacgc ataattttgt tgcctggtgg 1320catttgcaaa atgcataacc tatgcattta aaagattatg tatgctcttc tgacttttcg 1380tgtgatgagg ctcgtggaaa aaatgaataa tttatgaatt tgagaacaat tttgtgttgt 1440tacggtattt tactatggaa taatcaatca attgaggatt ttatgcaaat atcgtttgaa 1500tatttttccg accctttgag tacttttctt cataattgca taatattgtc cgctgcccgt 1560ttttctgtta gacggtgtct tgatctactt gctatcgttc aacaccacct tatcttctaa 1620ctattttttt tttagctcat ttgaatcagc ttatggtgat ggcacatttt tgcataaacc 1680tagctgtcct cgttgaacat aggaaaaaaa aatatataaa caaggctctt tcactctcct 1740tggaatcaga tttgggtttg ttccctttat tttcatattt cttgtcatat tcttttctca 1800attattattt tctactcata acctcacgca aaataacaca gtcaaatcaa tcaagtttaa 1860acagtatggc taaggaatac ttcccacaaa tccaaaagat ccaataccaa ggtccaaagt 1920ccactgaccc attgtccttc aagtactaca acccagaaga agtcatcaac ggtaaaacca 1980tgagagaaca cttgaagttc gctttgtctt ggtggcacac catgggtggt gacggtactg 2040atatgttcgg ttgtggtact actgacaaga cttggggtca atctgatcca gctgctagag 2100ctaaggctaa ggttgacgct gctttcgaaa tcatggacaa gttgtccatc gattactact 2160gtttccacga cagagatttg tctccagaat acggttcctt gaaggctacc aacgaccaat 2220tggatatcgt cactgactac atcaaggaaa agcaaggtga caagttcaag tgtttgtggg 2280gtactgctaa gtgtttcgac cacccaagat tcatgcacgg tgctggtact tctccatccg 2340ctgacgtttt cgctttctct gctgctcaaa tcaagaaggc tttggaatcc accgttaagt 2400tgggtgctaa cggttacgtc ttctggggtg gtagagaagg ttacgaaacc ttgttgaaca 2460ctaacatggg tttggaattg gacaacatgg ctagattgat gaagatggct gttgaatacg 2520gtagatctat cggtttcaag ggtgacttct acatcgaacc aaagccaaag gaaccaacta 2580agcaccaata cgacttcgat accgctactg tcttgggttt cttgagaaag tacggtttgg 2640acaaggattt caagatgaac atcgaagcta accacgctac cttggctcaa cacactttcc 2700aacacgaatt gagagttgct agagacaacg gtgtcttcgg ttccatcgat gctaaccaag 2760gtgacgtttt gttgggttgg gacaccgatc aattcccaac taacatctac gacaccacta 2820tgtgtatgta cgaagtcatc aaggctggtg gtttcaccaa cggtggtttg aacttcgacg 2880ctaaggctag aagaggttct ttcactccag aagacatctt ctactcctac atcgctggta 2940tggacgcttt cgctttgggt ttcagagctg ctttgaagtt gatcgaagac ggtagaatcg 3000ataagttcgt tgctgacaga tacgcttctt ggaacaccgg tatcggtgct gacatcatcg 3060ctggtaaagc tgatttcgct tctttggaaa agtacgcttt ggaaaagggt gaagtcactg 3120cttccttgtc ctctggtaga caagaaatgt tggaatccat cgtcaacaac gttttgttct 3180ctttgtgagg ccctgcaggc cagaggaaaa taatatcaag tgctggaaac tttttctctt 3240ggaatttttg caacatcaag tcatagtcaa ttgaattgac ccaatttcac atttaagatt 3300tttttttttt catccgacat acatctgtac actaggaagc cctgtttttc tgaagcagct 3360tcaaatatat atatttttta catatttatt atgattcaat gaacaatcta attaaatcga 3420aaacaagaac cgaaacgcga ataaataatt tatttagatg gtgacaagtg tataagtcct 3480catcgggaca gctacgattt ctctttcggt tttggctgag ctactggttg ctgtgacgca 3540gcggcattag cgcggcgtta tgagctaccc tcgtggcctg aaagatggcg ggaataaagc 3600ggaactaaaa attactgact gagccatatt gaggtcaatt tgtcaactcg tcaagtcacg 3660tttggtggac ggcccctttc caacgaatcg tatatactaa catgcgcgcg cttcctatat 3720acacatatac atatatatat atatatatat gtgtgcgtgt atgtgtacac ctgtatttaa 3780tttccttact cgcgggtttt tcttttttct caattcttgg cttcctcttt ctcgagcccg 3840ggatttaaat gccgttgaca ttcatgtaag cagtattagc ttttaaactg ccattaacgc 3900acctatacga ttgcggcggt gcgaaaacag taactagcac tcttacggct cattgtttcc 3960tctatccaaa gttgcctata tcacaataaa attagaaatt aatcatttta ataagcttgc 4020tgccattcag tgactcgcaa tctacgtttt aaacgtaatt taaaaatcca cttccaccta 4080catattttgc aacagtcgta cgacagaaac tgaggctcta taaaatgaga tgtgctaatt 4140gttctttctt ggctggtttc agctacatct ttctgtaatc aatctacaaa tttacacgcg 4200agcttcattt tgacagtaaa caaatgtaaa agacacatgc aataaaagcg gtccagaaaa 4260caaacgacaa agccaccaaa aatgtttgca aacgttggat ttagaacttt gagggggatc 4320cgcattgcgg attacgtatt ctaatgttca gtaccgttcg tataatgtat gctatacgaa 4380gttatgcaga ttgtactgag agtgcaccat accacagctt ttcaattcaa ttcatcattt 4440tttttttatt cttttttttg atttcggttt ctttgaaatt tttttgattc ggtaatctcc 4500gaacagaagg aagaacgaag gaaggagcac agacttagat tggtatatat acgcatatgt 4560agtgttgaag aaacatgaaa ttgcccagta ttcttaaccc aactgcacag aacaaaaacc 4620tgcaggaaac gaagataaat catgtcgaaa gctacatata aggaacgtgc tgctactcat 4680cctagtcctg ttgctgccaa gctatttaat atcatgcacg aaaagcaaac aaacttgtgt 4740gcttcattgg atgttcgtac caccaaggaa ttactggagt tagttgaagc attaggtccc 4800aaaatttgtt tactaaaaac acatgtggat atcttgactg atttttccat ggagggcaca 4860gttaagccgc taaaggcatt atccgccaag tacaattttt tactcttcga agacagaaaa 4920tttgctgaca ttggtaatac agtcaaattg cagtactctg cgggtgtata cagaatagca 4980gaatgggcag acattacgaa tgcacacggt gtggtgggcc caggtattgt tagcggtttg 5040aagcaggcgg cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt agcagaattg 5100tcatgcaagg gctccctatc tactggagaa tatactaagg gtactgttga cattgcgaag 5160agcgacaaag attttgttat cggctttatt gctcaaagag acatgggtgg aagagatgaa 5220ggttacgatt ggttgattat gacacccggt gtgggtttag atgacaaggg agacgcattg 5280ggtcaacagt atagaaccgt ggatgatgtg gtctctacag gatctgacat tattattgtt 5340ggaagaggac tatttgcaaa gggaagggat gctaaggtag agggtgaacg ttacagaaaa 5400gcaggctggg aagcatattt gagaagatgc ggccagcaaa actaaaaaac tgtattataa 5460gtaaatgcat gtatactaaa ctcacaaatt agagcttcaa tttaattata tcagttatta 5520ccctatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggaaatt 5580gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 5640aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 5700ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 5760aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 5820agataacttc gtataatgta tgctatacga acggtaccaa ttgtttctcc acatgtgtac 5880cgtataaagt tcttgttatg aattagaagg attatcacaa aagccctcca tcaaatctga 5940taatttcatt acccaaagac tgtaatacct tgggtgtaaa gatcgtattt tttgatgtct 6000tcgtgatatt ttgcagatct ttccatttta taaatttctg caaatatcgt gcatcagctc 6060atcttggaat tggcacaatc tgcgtaattt ttttcttggt tctcttctcc ttttgatttt 6120aagagcgctt tctattcagt agcagatttt cagtaaataa agccatcttt ctcaagtaaa 6180ctcatgatca tgcagttgtt caaaattaca ccaataacga tgttatcttt aaaactttcc 6240ccagaaagaa gatggcactt ttgcactaaa tacttcaaac tatttaactt tgaagcagga 6300catattgccg gggcgccgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 6360cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 6420agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 6480cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 6540actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 6600cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 6660accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 6720cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 6780tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 6840taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 6900tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 6960gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 7020gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 7080cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 7140tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 7200tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 7260atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 7320ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 7380gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 7440gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 7500gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 7560gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 7620gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 7680cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 7740atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 7800tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 7860ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 7920gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 7980tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 8040ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 8100ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 8160gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 8220ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 8280tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 8340ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 8400agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 8460agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 8520gggcggagcc tatggaa 8537506728DNAartificial sequenceconstructed vector 50acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 60aagtgccacc tgggtccttt tcatcacgtg ctataaaaat aattataatt taaatttttt 120aatataaata tataaattaa aaatagaaag taaaaaaaga aattaaagaa aaaatagttt 180ttgttttccg aagatgtaaa agactctagg gggatcgcca acaaatacta ccttttatct 240tgctcttcct gctctcaggt attaatgccg aattgtttca tcttgtctgt gtagaagacc 300acacacgaaa atcctgtgat tttacatttt acttatcgtt aatcgaatgt atatctattt 360aatctgcttt tcttgtctaa taaatatata tgtaaagtac gctttttgtt gaaatttttt 420aaacctttgt ttattttttt ttcttcattc cgtaactctt ctaccttctt tatttacttt 480ctaaaatcca aatacaaaac ataaaaataa ataaacacag agtaaattcc caaattattc 540catcattaaa agatacgagg cgcgtgtaag ttacaggcaa gcgatccgtc ctaagaaacc 600attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg 660cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct 720tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gcgtgttggc 780gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 840aaattcccgt tttaagagct tggtgagcgc taggagtcac tgccaggtat cgtttgaaca 900cggcattagt cagggaagtc ataacacagt cctttcccgc aattttcttt ttctattact 960cttggcctcc tctagtacac tctatatttt tttatgcctc ggtaatgatt ttcatttttt 1020tttttcccct agcggatgac tctttttttt tcttagcgat tggcattatc acataatgaa 1080ttatacatta tataaagtaa tgtgatttct tcgaagaata tactaaaaaa tgagcaggca 1140agataaacga aggcaaagat gacagagcag aaagccctag taaagcgtat tacaaatgaa 1200accaagattc agattgcgat ctctttaaag ggtggtcccc tagcgataga gcactcgatc 1260ttcccagaaa aagaggcaga agcagtagca gaacaggcca cacaatcgca agtgattaac 1320gtccacacag gtatagggtt tctggaccat atgatacatg ctctggccaa gcattccggc 1380tggtcgctaa tcgttgagtg cattggtgac ttacacatag acgaccatca caccactgaa 1440gactgcggga ttgctctcgg tcaagctttt aaagaggccc tactggcgcg tggagtaaaa 1500aggtttggat caggatttgc gcctttggat gaggcacttt ccagagcggt ggtagatctt 1560tcgaacaggc cgtacgcagt tgtcgaactt ggtttgcaaa gggagaaagt aggagatctc 1620tcttgcgaga tgatcccgca ttttcttgaa agctttgcag aggctagcag aattaccctc 1680cacgttgatt gtctgcgagg caagaatgat catcaccgta gtgagagtgc gttcaaggct 1740cttgcggttg ccataagaga agccacctcg cccaatggta ccaacgatgt tccctccacc 1800aaaggtgttc ttatgtagtg acaccgatta tttaaagctg cagcatacga tatatataca 1860tgtgtatata tgtataccta tgaatgtcag taagtatgta tacgaacagt atgatactga 1920agatgacaag gtaatgcatc attctatacg tgtcattctg aacgaggcgc gctttccttt 1980tttctttttg ctttttcttt ttttttctct tgaactcgac ggatctatgc ggtgtgaaat 2040accgcacaga tgcgtaagga gaaaataccg catcaggaaa ttgtaaacgt taatattttg 2100ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc 2160ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt 2220tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc 2280tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg 2340tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga 2400aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg 2460ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg 2520ctacagggcg cgtcgcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 2580gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 2640agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgagcgc 2700gcgtaatacg actcactata gggcgaattg ggtaccgggc cccccctcga ggtcgacggt 2760atcgataagc ttgattagaa gccgccgagc gggcgacagc cctccgacgg aagactctcc 2820tccgtgcgtc ctcgtcttca ccggtcgcgt tcctgaaacg cagatgtgcc tcgcgccgca 2880ctgctccgaa caataaagat tctacaatac tagcttttat ggttatgaag aggaaaaatt 2940ggcagtaacc tggccccaca aaccttcaaa ttaacgaatc aaattaacaa ccataggatg 3000ataatgcgat tagtttttta gccttatttc tggggtaatt aatcagcgaa gcgatgattt 3060ttgatctatt aacagatata taaatggaaa agctgcataa ccactttaac taatactttc 3120aacattttca gtttgtatta cttcttattc aaatgtcata aaagtatcaa caaaaaattg 3180ttaatatacc tctatacttt aacgtcaagg agaaaaatgt ccaatttact gcccgtacac 3240caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg 3300gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt 3360tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct 3420gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc 3480cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt 3540gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt 3600gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc 3660atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat 3720aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact 3780gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt 3840gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct 3900ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc 3960gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact 4020catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga 4080cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag 4140atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg 4200gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagga gtaagcgaat 4260ttcttatgat

ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 4320ttttaaagtg actcttaggt tttaaaacga aaattcttat tcttgagtaa ctctttcctg 4380taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 4440ggcatgccga gcaaatgcct gcaaatcgct ccccatttca cccaattgta gatatgctaa 4500ctccagcaat gagttgatga atctcggtgt gtattttatg tcctcagagg acaacacctg 4560tggtgttcta gagcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg 4620gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4680gctcacaatt ccacacaaca taggagccgg aagcataaag tgtaaagcct ggggtgccta 4740atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4800cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 4860tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4920agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4980aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 5040gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 5100tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 5160cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 5220ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 5280cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 5340atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 5400agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 5460gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 5520gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 5580tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 5640agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5700gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 5760aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 5820aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 5880ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 5940gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 6000aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 6060ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6120tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 6180ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 6240cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 6300agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 6360gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6420gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 6480acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 6540acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 6600agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 6660aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6720gagcggat 6728512431DNAartificial sequencechimeric expression cassette for BSaraA 51cttttctggc aaccaaaccc atacatcggg attcctataa taccttcgtt ggtctcccta 60acatgtaggt ggcggagggg agatatacaa tagaacagat accagacaag acataatggg 120ctaaacaaga ctacaccaat tacactgcct cattgatggt ggtacataac gaactaatac 180tgtagcccta gacttgatag ccatcatcat atcgaagttt cactaccctt tttccatttg 240ccatctattg aagtaataat aggcgcatgc aacttctttt cttttttttt cttttctctc 300tcccccgttg ttgtctcacc atatccgcaa tgacaaaaaa atgatggaag acactaaagg 360aaaaaattaa cgacaaagac agcaccaaca gatgtcgttg ttccagagct gatgaggggt 420atctcgaagc acacgaaact ttttccttcc ttcattcacg cacactactc tctaatgagc 480aacggtatac ggccttcctt ccagttactt gaatttgaaa taaaaaaaag tttgctgtct 540tgctatcaag tataaataga cctgcaatta ttaatctttt gtttcctcgt cattgttctc 600gttccctttc ttccttgttt ctttttctgc acaatatttc aagctatacc aagcatacaa 660tcaactatct catatacaat gttgcaaact aaggattacg aattctggtt cgttactggt 720tctcaacact tgtacggtga agaaactttg gaattggtcg atcaacacgc taagtctatc 780tgtgaaggtt tgtccggtgt ctcttccaga tacaagatca cccacaagcc agttgtcacc 840tcttccgaaa ctatcagaca attgttgaga gaagctgaat actctgaaac ttgtgctggt 900atcatcacct ggatgcacac tttctctcca gctaagatgt ggatcgaagg tttgtcttcc 960taccaaaagc cattgatgca cttgcacacc caatacaaca gagacatccc ttggggtact 1020atcgacatgg atttcatgaa ctctaaccaa tccgctcacg gtgacagaga atacggttac 1080atcaactcca gaatgggttt gtccagaaag gttgtcgctg gttactggga cgatgaagaa 1140gtcaagaagg aaatctctca atggatggac accgctgctg ctttgaacga atccagacac 1200atcaaggttg ctagattcgg tgacaacatg agacacgttg ctgtcactga cggtgacaag 1260gttggtgctc acatccaatt cggttggcaa gttgacggtt acggtatcgg tgacttggtt 1320gaagtcatga acagaatcac cgacgatgaa gttgacactt tgtacgctga atacgataga 1380ttgtacgtca tctctgaaga aaccaagaga gacgaagcta aggttgcttc catcaaggaa 1440caagctaaga tcgaattggg tttgaccact ttcttggaac aaggtggtta ctctgctttc 1500accacttcct tcgaagtctt gcacggtatg aagcaattgc caggtttggc tgttcaaaga 1560ttgatggaaa agggttacgg tttcgctggt gaaggtgact ggaagaccgc tgctttggtc 1620agaatgatga agatcatgtc tcaaggtaaa agaacctcct tcatggaaga ctacacttac 1680cacttcgaac caggtaacga aatgatcttg ggttctcaca tgttggaagt ttgtccaact 1740gtcgctttgg accaaccaaa gatcgaagtt cacccattgt ctatcggtgg taaagaagat 1800ccagctagat tcgtcttcaa cggtatctct ggttccgcta tccaagcctc tttggttgac 1860atcggtggta gattcagatt ggttttgaac gaagtcaacg gtcaagaaat cgaaaaggac 1920atgccaaact tgccagttgc tagagtcttg tggaagccag aaccatcttt gaagactgct 1980gctgaagcct ggatcttggc tggtggtgct caccacacct gtttgtctta cgaattgact 2040gtcgaacaaa tgttggactg ggctgaaatg gctggtatcg aatctgtttt gatctccaga 2100gataccacta tccacaagtt gaagcacgaa ttgaagtgga acacgaagcc ttgtacagat 2160tgcaaaagta attaattaat catgtaatta gttatgtcac gcttacattc acgccctcct 2220cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta ggtccctatt 2280tatttttttt aatagttatg ttagtattaa gaacgttatt tatatttcaa atttttcttt 2340tttttctgta caaacgcgtg tacgcatgta acattatact gaaaaccttg cttgagaagg 2400ttttgggacg ctcgaaggct ttaatttgcg g 243152566PRTEscherichia coli 52Met Ala Ile Ala Ile Gly Leu Asp Phe Gly Ser Asp Ser Val Arg Ala1 5 10 15Leu Ala Val Asp Cys Ala Ser Gly Glu Glu Ile Ala Thr Ser Val Glu 20 25 30Trp Tyr Pro Arg Trp Gln Lys Gly Gln Phe Cys Asp Ala Pro Asn Asn 35 40 45Gln Phe Arg His His Pro Arg Asp Tyr Ile Glu Ser Met Glu Ala Ala 50 55 60Leu Lys Thr Val Leu Ala Glu Leu Ser Val Glu Gln Arg Ala Ala Val65 70 75 80Val Gly Ile Gly Val Asp Ser Thr Gly Ser Thr Pro Ala Pro Ile Asp 85 90 95Ala Asp Gly Asn Val Leu Ala Leu Arg Pro Glu Phe Ala Glu Asn Pro 100 105 110Asn Ala Met Phe Val Leu Trp Lys Asp His Thr Ala Val Glu Arg Ser 115 120 125Glu Glu Ile Thr Arg Leu Cys His Ala Pro Gly Asn Val Asp Tyr Ser 130 135 140Arg Tyr Ile Gly Gly Ile Tyr Ser Ser Glu Trp Phe Trp Ala Lys Ile145 150 155 160Leu His Val Thr Arg Gln Asp Ser Ala Val Ala Gln Ser Ala Ala Ser 165 170 175Trp Ile Glu Leu Cys Asp Trp Val Pro Ala Leu Leu Ser Gly Thr Thr 180 185 190Arg Pro Gln Asp Ile Arg Arg Gly Arg Cys Ser Ala Gly His Lys Ser 195 200 205Leu Trp His Glu Ser Trp Gly Gly Leu Pro Pro Ala Ser Phe Phe Asp 210 215 220Glu Leu Asp Pro Ile Leu Asn Arg His Leu Pro Ser Pro Leu Phe Thr225 230 235 240Asp Thr Trp Thr Ala Asp Ile Pro Val Gly Thr Leu Cys Pro Glu Trp 245 250 255Ala Gln Arg Leu Gly Leu Pro Glu Ser Val Val Ile Ser Gly Gly Ala 260 265 270Phe Asp Cys His Met Gly Ala Val Gly Ala Gly Ala Gln Pro Asn Ala 275 280 285Leu Val Lys Val Ile Gly Thr Ser Thr Cys Asp Ile Leu Ile Ala Asp 290 295 300Lys Gln Ser Val Gly Glu Arg Ala Val Lys Gly Ile Cys Gly Gln Val305 310 315 320Asp Gly Ser Val Val Pro Gly Phe Ile Gly Leu Glu Ala Gly Gln Ser 325 330 335Ala Phe Gly Asp Ile Tyr Ala Trp Phe Gly Arg Val Leu Ser Trp Pro 340 345 350Leu Glu Gln Leu Ala Ala Gln His Pro Glu Leu Lys Ala Gln Ile Asn 355 360 365Ala Ser Gln Lys Gln Leu Leu Pro Ala Leu Thr Glu Ala Trp Ala Lys 370 375 380Asn Pro Ser Leu Asp His Leu Pro Val Val Leu Asp Trp Phe Asn Gly385 390 395 400Arg Arg Ser Pro Asn Ala Asn Gln Arg Leu Lys Gly Val Ile Thr Asp 405 410 415Leu Asn Leu Ala Thr Asp Ala Pro Leu Leu Phe Gly Gly Leu Ile Ala 420 425 430Ala Thr Ala Phe Gly Ala Arg Ala Ile Met Glu Cys Phe Thr Asp Gln 435 440 445Gly Ile Ala Val Asn Asn Val Met Ala Leu Gly Gly Ile Ala Arg Lys 450 455 460Asn Gln Val Ile Met Gln Ala Cys Cys Asp Val Leu Asn Arg Pro Leu465 470 475 480Gln Ile Val Ala Ser Asp Gln Cys Cys Ala Leu Gly Ala Ala Ile Phe 485 490 495Ala Ala Val Ala Ala Lys Val His Ala Asp Ile Pro Ser Ala Gln Gln 500 505 510Lys Met Ala Ser Ala Val Glu Lys Thr Leu Gln Pro Arg Ser Glu Gln 515 520 525Ala Gln Arg Phe Glu Gln Leu Tyr Arg Arg Tyr Gln Gln Trp Ala Met 530 535 540Ser Ala Glu Gln His Tyr Leu Pro Thr Ser Ala Pro Ala Gln Ala Ala545 550 555 560Gln Ala Val Ala Thr Leu 565531701DNAartificial sequencecodon optimized coding region for E. coli ribulokinase 53atggctatcg ctatcggttt ggacttcggt tctgactccg ttagagcttt ggctgttgac 60tgtgcttccg gtgaagaaat cgctacttct gtcgaatggt atccaagatg gcaaaagggt 120caattctgtg acgctccaaa caaccaattc agacaccacc caagagatta catcgaatct 180atggaagctg ctttgaagac tgttttggct gaattgtctg tcgaacaaag agctgctgtt 240gtcggtatcg gtgttgactc tactggttcc accccagctc caatcgacgc tgatggtaac 300gttttggctt tgagaccaga attcgctgaa aacccaaacg ctatgttcgt tttgtggaag 360gaccacactg ctgtcgaaag atccgaagaa atcaccagat tgtgtcacgc tccaggtaac 420gttgactact ccagatacat cggtggtatc tactcttccg aatggttctg ggctaagatt 480ttgcacgtta ctagacaaga ctctgctgtc gctcaatctg ctgcttcctg gatcgaattg 540tgtgactggg ttccagcttt gttgtctggt actactagac cacaagatat cagaagaggt 600agatgttctg ctggtcacaa gtccttgtgg cacgaatctt ggggtggttt gccaccagct 660tccttcttcg acgaattgga cccaatcttg aacagacact tgccatctcc attgttcacc 720gacacttgga ccgctgatat cccagtcggt actttgtgtc cagaatgggc tcaaagattg 780ggtttgccag aatctgttgt catctccggt ggtgctttcg actgtcacat gggtgctgtt 840ggtgctggtg ctcaaccaaa cgctttggtt aaggtcatcg gtacttccac ctgtgacatc 900ttgatcgctg ataagcaatc tgttggtgaa agagctgtca agggtatctg tggtcaagtt 960gacggttccg ttgtcccagg tttcatcggt ttggaagctg gtcaatctgc tttcggtgac 1020atctacgctt ggttcggtag agttttgtcc tggccattgg aacaattggc tgctcaacac 1080ccagaattga aggctcaaat caacgcttct caaaagcaat tgttgccagc tttgactgaa 1140gcctgggcta agaacccatc cttggaccac ttgccagttg tcttggattg gttcaacggt 1200agaagatccc caaacgctaa ccaaagattg aagggtgtta tcactgactt gaacttggct 1260accgatgctc cattgttgtt cggtggtttg atcgctgcta ctgctttcgg tgctagagct 1320atcatggaat gtttcaccga ccaaggtatc gctgttaaca acgtcatggc tttgggtggt 1380atcgctagaa agaaccaagt tatcatgcaa gcctgttgtg acgtcttgaa cagaccattg 1440caaatcgttg cttctgatca atgttgtgct ttgggtgctg ctatcttcgc tgctgttgct 1500gctaaggtcc acgctgacat cccatccgct caacaaaaga tggcttctgc tgtcgaaaag 1560accttgcaac caagatccga acaagctcaa agattcgaac aattgtacag aagataccaa 1620caatgggcta tgtccgctga acaacactac ttgccaactt ctgctccagc tcaagctgct 1680caagctgttg ctaccttgta a 1701542911DNAartificial sequenceconstructed chimeric expression cassette for ECaraB 54gagaagaggt atacataaca agaaaatcgc gtgaacacct tatataactt agcccgttat 60tgagctaaaa aaccttgcaa aatttcctat gaataagaat acttcagacg tgataaaaat 120ttactttcta actcttctca cgctgcccct atctgttctt ccgctctacc gtgagaaata 180aagcatcgag tacggcagtt cgctgtcact gaactaaaac aataaggcta gttcgaatga 240tgaacttgct tgctgtcaaa cttctgagtt gccgctgatg tgacactgtg acaataaatt 300caaaccggtt atagcggtct cctccggtac cggttctgcc acctccaata gagctcagta 360ggagtcagaa cctctgcggt ggctgtcagt gactcatccg cgtttcgtaa gttgtgcgcg 420tgcacatttc gcccgttccc gctcatcttg cagcaggcga aattttcatc acgctgtagg 480acgcaaaaaa aaaataatta atcgtacaag aatcttggaa aaaaaattga aaaattttgt 540ataaaaggga tgacctaact tgactcaatg gcttttacac ccagtatttt ccctttcctt 600gtttgttaca attatagaag caagacaaaa acatatagac aacctattcc taggagttat 660atttttttac cctaccagca atataagtaa aaaataaaac atggctatcg ctatcggttt 720ggacttcggt tctgactccg ttagagcttt ggctgttgac tgtgcttccg gtgaagaaat 780cgctacttct gtcgaatggt atccaagatg gcaaaagggt caattctgtg acgctccaaa 840caaccaattc agacaccacc caagagatta catcgaatct atggaagctg ctttgaagac 900tgttttggct gaattgtctg tcgaacaaag agctgctgtt gtcggtatcg gtgttgactc 960tactggttcc accccagctc caatcgacgc tgatggtaac gttttggctt tgagaccaga 1020attcgctgaa aacccaaacg ctatgttcgt tttgtggaag gaccacactg ctgtcgaaag 1080atccgaagaa atcaccagat tgtgtcacgc tccaggtaac gttgactact ccagatacat 1140cggtggtatc tactcttccg aatggttctg ggctaagatt ttgcacgtta ctagacaaga 1200ctctgctgtc gctcaatctg ctgcttcctg gatcgaattg tgtgactggg ttccagcttt 1260gttgtctggt actactagac cacaagatat cagaagaggt agatgttctg ctggtcacaa 1320gtccttgtgg cacgaatctt ggggtggttt gccaccagct tccttcttcg acgaattgga 1380cccaatcttg aacagacact tgccatctcc attgttcacc gacacttgga ccgctgatat 1440cccagtcggt actttgtgtc cagaatgggc tcaaagattg ggtttgccag aatctgttgt 1500catctccggt ggtgctttcg actgtcacat gggtgctgtt ggtgctggtg ctcaaccaaa 1560cgctttggtt aaggtcatcg gtacttccac ctgtgacatc ttgatcgctg ataagcaatc 1620tgttggtgaa agagctgtca agggtatctg tggtcaagtt gacggttccg ttgtcccagg 1680tttcatcggt ttggaagctg gtcaatctgc tttcggtgac atctacgctt ggttcggtag 1740agttttgtcc tggccattgg aacaattggc tgctcaacac ccagaattga aggctcaaat 1800caacgcttct caaaagcaat tgttgccagc tttgactgaa gcctgggcta agaacccatc 1860cttggaccac ttgccagttg tcttggattg gttcaacggt agaagatccc caaacgctaa 1920ccaaagattg aagggtgtta tcactgactt gaacttggct accgatgctc cattgttgtt 1980cggtggtttg atcgctgcta ctgctttcgg tgctagagct atcatggaat gtttcaccga 2040ccaaggtatc gctgttaaca acgtcatggc tttgggtggt atcgctagaa agaaccaagt 2100tatcatgcaa gcctgttgtg acgtcttgaa cagaccattg caaatcgttg cttctgatca 2160atgttgtgct ttgggtgctg ctatcttcgc tgctgttgct gctaaggtcc acgctgacat 2220cccatccgct caacaaaaga tggcttctgc tgtcgaaaag accttgcaac caagatccga 2280acaagctcaa agattcgaac aattgtacag aagataccaa caatgggcta tgtccgctga 2340acaacactac ttgccaactt ctbamhgctc cagctcaagc tgctcaagct gttgctacct 2400tgtaaggatc caggagcaat gcaaaatcta ggggtagaat tactttttga aaaggaaaaa 2460tattcaggtt tgttgttttt atgtaagttg tatgatttga tatacatata tatatatata 2520taatatatat tgtacatgtg tttttccggg gaagaatgga ttatccggag gtgtgaataa 2580aatgatgacg attataggtt tgtgttgtaa tatttagata actcaattct cgccagtttg 2640aactccaacc tagactggtt caaagctttt gctatcaaga tgagatatat ggaattttcg 2700tctttatcgt ccacttgtat ctttatttcc tcgtcatctt catcaatatt gattccatta 2760ataatcgatt tatcgctcag agtgttgacc aattcggtct tgttggggaa gaaatgttcc 2820atttttcttc ccaagttttg aattctttca caaacccagg caattctttg taagcctaat 2880gcagcagaag aaccctttaa aaaatggccc a 291155231PRTEscherichia coli 55Met Leu Glu Asp Leu Lys Arg Gln Val Leu Glu Ala Asn Leu Ala Leu1 5 10 15Pro Lys His Asn Leu Val Thr Leu Thr Trp Gly Asn Val Ser Ala Val 20 25 30Asp Arg Glu Arg Gly Val Phe Val Ile Lys Pro Ser Gly Val Asp Tyr 35 40 45Ser Ile Met Thr Ala Asp Asp Met Val Val Val Ser Ile Glu Thr Gly 50 55 60Glu Val Val Glu Gly Ala Lys Lys Pro Ser Ser Asp Thr Pro Thr His65 70 75 80Arg Leu Leu Tyr Gln Ala Phe Pro Ser Ile Gly Gly Ile Val His Thr 85 90 95His Ser Arg His Ala Thr Ile Trp Ala Gln Ala Gly Gln Ser Ile Pro 100 105 110Ala Thr Gly Thr Thr His Ala Asp Tyr Phe Tyr Gly Thr Ile Pro Cys 115 120 125Thr Arg Lys Met Thr Asp Ala Glu Ile Asn Gly Glu Tyr Glu Trp Glu 130 135 140Thr Gly Asn Val Ile Val Glu Thr Phe Glu Lys Gln Gly Ile Asp Ala145 150 155 160Ala Gln Met Pro Gly Val Leu Val His Ser His Gly Pro Phe Ala Trp 165 170 175Gly Lys Asn Ala Glu Asp Ala Val His Asn Ala Ile Val Leu Glu Glu 180 185 190Val Ala Tyr Met Gly Ile Phe Cys Arg Gln Leu Ala Pro Gln Leu Pro 195 200 205Asp Met Gln Gln Thr Leu Leu Asn Lys His Tyr Leu Arg Lys His Gly 210 215 220Ala Lys Ala Tyr Tyr Gly Gln225 23056696DNAartificial sequenceE coli L-ribulose-5-phosphate 4-epimerase codon optimized coding region 56atgttggaag atttgaagag acaagttttg gaagctaact tggctttgcc aaagcacaac 60ttggtcacct tgacctgggg taacgtctct gctgttgaca gagaaagagg tgtcttcgtt 120atcaagccat ctggtgttga ttactccatc atgactgctg acgatatggt tgtcgtttcc 180atcgaaaccg gtgaagtcgt tgaaggtgct aagaagccat cttccgacac

cccaactcac 240agattgttgt accaagcctt cccatctatc ggtggtatcg tccacactca ctccagacac 300gctaccatct gggctcaagc tggtcaatct atcccagcta ctggtactac tcacgctgac 360tacttctacg gtactatccc atgtactaga aagatgaccg atgctgaaat caacggtgaa 420tacgaatggg aaactggtaa cgtcatcgtt gaaaccttcg aaaagcaagg tatcgacgct 480gctcaaatgc caggtgtctt ggttcactct cacggtccat tcgcttgggg taaaaacgct 540gaagatgctg ttcacaacgc tatcgtcttg gaagaagttg cttacatggg tatcttctgt 600agacaattgg ctccacaatt gccagacatg caacaaacct tgttgaacaa gcactacttg 660agaaagcacg gtgctaaggc ttactacggt caataa 696571691DNAartificial sequenceconstructed chimeric expression cassette for ECaraD 57agttcgagtt tatcattatc aatactgcca tttcaaagaa tacgtaaata attaatagta 60gtgattttcc taactttatt tagtcaaaaa attagccttt taattctgct gtaacccgta 120catgcccaaa atagggggcg ggttacacag aatatataac atcgtaggtg tctgggtgaa 180cagtttattc ctggcatcca ctaaatataa tggagcccgc tttttaagct ggcatccaga 240aaaaaaaaga atcccagcac caaaatattg ttttcttcac caaccatcag ttcataggtc 300cattctctta gcgcaactac agagaacagg ggcacaaaca ggcaaaaaac gggcacaacc 360tcaatggagt gatgcaacct gcctggagta aatgatgaca caaggcaatt gacccacgca 420tgtatctatc tcattttctt acaccttcta ttaccttctg ctctctctga tttggaaaaa 480gctgaaaaaa aaggttgaaa ccagttccct gaaattattc ccctacttga ctaataagta 540tataaagacg gtaggtattg attgtaattc tgtaaatcta tttcttaaac ttcttaaatt 600ctacttttat agttagtctt ttttttagtt ttaaaacacc aagaacttag tttcgaataa 660acacacataa acaaacaaaa tgttggaaga tttgaagaga caagttttgg aagctaactt 720ggctttgcca aagcacaact tggtcacctt gacctggggt aacgtctctg ctgttgacag 780agaaagaggt gtcttcgtta tcaagccatc tggtgttgat tactccatca tgactgctga 840cgatatggtt gtcgtttcca tcgaaaccgg tgaagtcgtt gaaggtgcta agaagccatc 900ttccgacacc ccaactcaca gattgttgta ccaagccttc ccatctatcg gtggtatcgt 960ccacactcac tccagacacg ctaccatctg ggctcaagct ggtcaatcta tcccagctac 1020tggtactact cacgctgact acttctacgg tactatccca tgtactagaa agatgaccga 1080tgctgaaatc aacggtgaat acgaatggga aactggtaac gtcatcgttg aaaccttcga 1140aaagcaaggt atcgacgctg ctcaaatgcc aggtgtcttg gttcactctc acggtccatt 1200cgcttggggt aaaaacgctg aagatgctgt tcacaacgct atcgtcttgg aagaagttgc 1260ttacatgggt atcttctgta gacaattggc tccacaattg ccagacatgc aacaaacctt 1320gttgaacaag cactacttga gaaagcacgg tgctaaggct tactacggtc aataagagta 1380agcgaatttc ttatgattta tgatttttat tattaaataa gttataaaaa aaataagtgt 1440atacaaattt taaagtgact cttaggtttt aaaacgaaaa ttcttattct tgagtaactc 1500tttcctgtag gtcaggttgc tttctcaggt atagcatgag gtcgctctta ttgaccacac 1560ctctaccggc atgccgagca aatgcctgca aatcgctccc catttcaccc aattgtagat 1620atgctaactc cagcaatgag ttgatgaatc tcggtgtgta ttttatgtcc tcagaggaca 1680acacctgtgg t 1691588202DNAartificial sequenceconstructed plasmidmisc_feature(421)..(421)n is a, c, g, or t 58tcccattacc gacatttggg cgctatacgt gcatatgttc atgtatgtat ctgtatttaa 60aacacttttg tattattttt cctcatatat gtgtataggt ttatacggat gatttaatta 120ttacttcacc accctttatt tcaggctgat atcttagcct tgttactaga ttaatcatgt 180aattagttat gtcacgctta cattcacgcc ctccccccac atccgctcta accgaaaagg 240aaggagttag acaacctgaa gtctaggtcc ctatttattt ttttatagtt atgttagtat 300taagaacgtt atttatattt caaatttttc ttttttttct gtacagacgc gtgtacgcat 360gtaacattat actgaaaacc ttgcttgaga aggttttggg acgctcgaag gctttaattt 420ntgcgggcgg ccgctggaca atttattcat ggcatcgtca ttgatataag tggcttgagc 480tgtggataag aaaagccata tatttatata aacatttaga tatgaatagg aagtagattg 540ttcgacgcaa ctacccgttc aagaagtata atggggaatg gtctcatctt ccctcacagg 600atatagttct ctgaagagat acatacgttt gtgtatacta tgcttcttta tcaactcaag 660ttttgtagag gaagacgttg aagatggtga tgtgacatct ttactattct ccagcacgtt 720ttcagtattt acttaatcgt atattaatga cgtcccttat ctattaactt tccggttttt 780ctttttttcg gtgaatgttc tttccgtttt agtgaatttt tcaattgtaa ttgacgcaat 840cggtttataa caagcagaca taaatatcaa gctcgagcca aatcacaaaa aaagccttat 900agbamhcttg ccctgacaaa gaatatacaa ctcgggaagg atccgagaag aggtatacat 960aacaagaaaa tcgcgtgaac accttatata acttagcccg ttattgagct aaaaaacctt 1020gcaaaatttc ctatgaataa gaatacttca gacgtgataa aaatttactt tctaactctt 1080ctcacgctgc ccctatctgt tcttccgctc taccgtgaga aataaagcat cgagtacggc 1140agttcgctgt cactgaacta aaacaataag gctagttcga atgatgaact tgcttgctgt 1200caaacttctg agttgccgct gatgtgacac tgtgacaata aattcaaacc ggttatagcg 1260gtctcctccg gtaccggttc tgccacctcc aatagagctc agtaggagtc agaacctctg 1320cggtggctgt cagtgactca tccgcgtttc gtaagttgtg cgcgtgcaca tttcgcccgt 1380tcccgctcat cttgcagcag gcgaaatttt catcacgctg taggacgcaa aaaaaaaata 1440attaatcgta caagaatctt ggaaaaaaaa ttgaaaaatt ttgtataaaa gggatgacct 1500aacttgactc aatggctttt acacccagta ttttcccttt ccttgtttgt tacaattata 1560gaagcaagac aaaaacatat agacaaccta ttcctaggag ttatattttt ttaccctacc 1620agcaatataa gtaaaaaata aaacatggct atcgctatcg gtttggactt cggttctgac 1680tccgttagag ctttggctgt tgactgtgct tccggtgaag aaatcgctac ttctgtcgaa 1740tggtatccaa gatggcaaaa gggtcaattc tgtgacgctc caaacaacca attcagacac 1800cacccaagag attacatcga atctatggaa gctgctttga agactgtttt ggctgaattg 1860tctgtcgaac aaagagctgc tgttgtcggt atcggtgttg actctactgg ttccacccca 1920gctccaatcg acgctgatgg taacgttttg gctttgagac cagaattcgc tgaaaaccca 1980aacgctatgt tcgttttgtg gaaggaccac actgctgtcg aaagatccga agaaatcacc 2040agattgtgtc acgctccagg taacgttgac tactccagat acatcggtgg tatctactct 2100tccgaatggt tctgggctaa gattttgcac gttactagac aagactctgc tgtcgctcaa 2160tctgctgctt cctggatcga attgtgtgac tgggttccag ctttgttgtc tggtactact 2220agaccacaag atatcagaag aggtagatgt tctgctggtc acaagtcctt gtggcacgaa 2280tcttggggtg gtttgccacc agcttccttc ttcgacgaat tggacccaat cttgaacaga 2340cacttgccat ctccattgtt caccgacact tggaccgctg atatcccagt cggtactttg 2400tgtccagaat gggctcaaag attgggtttg ccagaatctg ttgtcatctc cggtggtgct 2460ttcgactgtc acatgggtgc tgttggtgct ggtgctcaac caaacgcttt ggttaaggtc 2520atcggtactt ccacctgtga catcttgatc gctgataagc aatctgttgg tgaaagagct 2580gtcaagggta tctgtggtca agttgacggt tccgttgtcc caggtttcat cggtttggaa 2640gctggtcaat ctgctttcgg tgacatctac gcttggttcg gtagagtttt gtcctggcca 2700ttggaacaat tggctgctca acacccagaa ttgaaggctc aaatcaacgc ttctcaaaag 2760caattgttgc cagctttgac tgaagcctgg gctaagaacc catccttgga ccacttgcca 2820gttgtcttgg attggttcaa cggtagaaga tccccaaacg ctaaccaaag attgaagggt 2880gttatcactg acttgaactt ggctaccgat gctccattgt tgttcggtgg tttgatcgct 2940gctactgctt tcggtgctag agctatcatg gaatgtttca ccgaccaagg tatcgctgtt 3000aacaacgtca tggctttggg tggtatcgct agaaagaacc aagttatcat gcaagcctgt 3060tgtgacgtct tgaacagacc attgcaaatc gttgcttctg atcaatgttg tgctttgggt 3120gctgctatct tcgctgctgt tgctgctaag gtccacgctg acatcccatc cgctcaacaa 3180aagatggctt ctgctgtcga aaagaccttg caaccaagat ccgaacaagc tcaaagattc 3240gaacaattgt acagaagata ccaacaatgg gctatgtccg ctgaacaaca ctacttgcca 3300acttctbamh gctccagctc aagctgctca agctgttgct accttgtaag gatccaggag 3360caatgcaaaa tctaggggta gaattacttt ttgaaaagga aaaatattca ggtttgttgt 3420ttttatgtaa gttgtatgat ttgatataca tatatatata tatataatat atattgtaca 3480tgtgtttttc cggggaagaa tggattatcc ggaggtgtga ataaaatgat gacgattata 3540ggtttgtgtt gtaatattta gataactcaa ttctcgccag tttgaactcc aacctagact 3600ggttcaaagc ttttgctatc aagatgagat atatggaatt ttcgtcttta tcgtccactt 3660gtatctttat ttcctcgtca tcttcatcaa tattgattcc attaataatc gatttatcgc 3720tcagagtgtt gaccaattcg gtcttgttgg ggaagaaatg ttccattttt cttcccaagt 3780tttgaattct ttcacaaacc caggcaattc tttgtaagcc taatgcagca gaagaaccct 3840ttaaaaaatg gcccattaat gtggctgtgg tttcagggtc cataaagctt ttcaattcat 3900cttttttttt tttgttcttt tttttgattc cggtttcttt gaaatttttt tgattcggta 3960atctccgagc agaaggaaga acgaaggaag gagcacagac ttagattggt atatatacgc 4020atatgtggtg ttgaagaaac atgaaattgc ccagtattct taacccaact gcacagaaca 4080aaaacctgca ggaaacgaag ataaatcatg tcgaaagcta catataagga acgtgctgct 4140actcatccta gtcctgttgc tgccaagcta tttaatatca tgcacgaaaa gcaaacaaac 4200ttgtgtgctt cattggatgt tcgtaccacc aaggaattac tggagttagt tgaagcatta 4260ggtcccaaaa tttgtttact aaaaacacat gtggatatct tgactgattt ttccatggag 4320ggcacagtta agccgctaaa ggcattatcc gccaagtaca attttttact cttcgaagac 4380agaaaatttg ctgacattgg taatacagtc aaattgcagt actctgcggg tgtatacaga 4440atagcagaat gggcagacat tacgaatgca cacggtgtgg tgggcccagg tattgttagc 4500ggtttgaagc aggcggcgga agaagtaaca aaggaaccta gaggcctttt gatgttagca 4560gaattgtcat gcaagggctc cctagctact ggagaatata ctaagggtac tgttgacatt 4620gcgaagagcg acaaagattt tgttatcggc tttattgctc aaagagacat gggtggaaga 4680gatgaaggtt acgattggtt gattatgaca cccggtgtgg gtttagatga caagggagac 4740gcattgggtc aacagtatag aaccgtggat gatgtggtct ctacaggatc tgacattatt 4800attgttggaa gaggactatt tgcaaaggga agggatgcta aggtagaggg tgaacgttac 4860agaaaagcag gctgggaagc atatttgaga agatgcggcc agcaaaacta aaaaactgta 4920ttataagtaa atgcatgtat actaaactca caaattagag cttcaattta attatatcag 4980ttattacccg ggaatctcgg tcgtaatgat ttctataatg acgaaaaaaa aaaaattgga 5040aagaaaaagc ttcatggcct tctaaatcac catttttggt gaacggcctt gataaggatg 5100ttagttgtgt tattgctggg ttagacacga aggtaaatta ccaccgtttg gctgttacac 5160tgcagtattt gcagaaggat tctgttcact ttgttggtac aaatgttgat tctactttcc 5220cgcaaaaggg ttatacattt cccggtgcag gctccatgat tgaatcattg gcattctcat 5280ctaataggag gccatcgtac tgtggtaagc caaatcaaaa tatgctaaac agcattatat 5340cggcattcaa cctggataga tcaaagtgct gtatggttgg tgacagatta aacaccgata 5400tgaaattcgg tgttgaaggt gggttaggtg gcacactact cgttttgagt ggtattgaaa 5460ccgaagagag agccttgaag atttcgcacg attatccaag acctaaattt tacattgata 5520aacttggtga asccatctac accttaacca ataatgagtt atagggcgcg ccgacgtcag 5580gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 5640caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 5700ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 5760gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 5820tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 5880ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 5940tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 6000atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 6060gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 6120caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 6180ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 6240ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 6300ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 6360ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 6420gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 6480ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 6540taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 6600agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 6660atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 6720aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 6780caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 6840ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 6900cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 6960tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 7020gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 7080ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 7140gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 7200caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 7260ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 7320tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 7380ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 7440agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 7500aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 7560gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 7620tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 7680tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 7740ccaagctttt tctttccaat tttttttttt tcgtcattat aaaaatcatt acgaccgaga 7800ttccctaata agagaatagg aacttcggaa taggaacttc aaagcgtttc cgaaaacgag 7860cgcttccgaa aatgcaacgc gagctgcgca catacagctc actgttcacg tcgcacctat 7920atctgcgtgt tgcctgtata tatatataca tgagaagaac ggcatagtgc gtgtttatgc 7980ttaaatgcgt acttatatgc gtctatttat gtaggatgaa aggtagtcta gtacctcctg 8040tgatattatc ccattccatg cggggtatcg tatgcttcct tcagcactac cctttagctg 8100ttctatatgc tgccactcct caattggatt agtctcatcc ttcaatgcta tcatttcctt 8160tgatattgga tcatatgcat agtaccgaga aactagagga tc 82025910887DNAartificial sequenceconstructed plasmid 59ggcctcgagc ggaccggatc cttttctggc aaccaaaccc atacatcggg attcctataa 60taccttcgtt ggtctcccta acatgtaggt ggcggagggg agatatacaa tagaacagat 120accagacaag acataatggg ctaaacaaga ctacaccaat tacactgcct cattgatggt 180ggtacataac gaactaatac tgtagcccta gacttgatag ccatcatcat atcgaagttt 240cactaccctt tttccatttg ccatctattg aagtaataat aggcgcatgc aacttctttt 300cttttttttt cttttctctc tcccccgttg ttgtctcacc atatccgcaa tgacaaaaaa 360atgatggaag acactaaagg aaaaaattaa cgacaaagac agcaccaaca gatgtcgttg 420ttccagagct gatgaggggt atctcgaagc acacgaaact ttttccttcc ttcattcacg 480cacactactc tctaatgagc aacggtatac ggccttcctt ccagttactt gaatttgaaa 540taaaaaaaag tttgctgtct tgctatcaag tataaataga cctgcaatta ttaatctttt 600gtttcctcgt cattgttctc gttccctttc ttccttgttt ctttttctgc acaatatttc 660aagctatacc aagcatacaa tcaactatct catatacaat gttgcaaact aaggattacg 720aattctggtt cgttactggt tctcaacact tgtacggtga agaaactttg gaattggtcg 780atcaacacgc taagtctatc tgtgaaggtt tgtccggtgt ctcttccaga tacaagatca 840cccacaagcc agttgtcacc tcttccgaaa ctatcagaca attgttgaga gaagctgaat 900actctgaaac ttgtgctggt atcatcacct ggatgcacac tttctctcca gctaagatgt 960ggatcgaagg tttgtcttcc taccaaaagc cattgatgca cttgcacacc caatacaaca 1020gagacatccc ttggggtact atcgacatgg atttcatgaa ctctaaccaa tccgctcacg 1080gtgacagaga atacggttac atcaactcca gaatgggttt gtccagaaag gttgtcgctg 1140gttactggga cgatgaagaa gtcaagaagg aaatctctca atggatggac accgctgctg 1200ctttgaacga atccagacac atcaaggttg ctagattcgg tgacaacatg agacacgttg 1260ctgtcactga cggtgacaag gttggtgctc acatccaatt cggttggcaa gttgacggtt 1320acggtatcgg tgacttggtt gaagtcatga acagaatcac cgacgatgaa gttgacactt 1380tgtacgctga atacgataga ttgtacgtca tctctgaaga aaccaagaga gacgaagcta 1440aggttgcttc catcaaggaa caagctaaga tcgaattggg tttgaccact ttcttggaac 1500aaggtggtta ctctgctttc accacttcct tcgaagtctt gcacggtatg aagcaattgc 1560caggtttggc tgttcaaaga ttgatggaaa agggttacgg tttcgctggt gaaggtgact 1620ggaagaccgc tgctttggtc agaatgatga agatcatgtc tcaaggtaaa agaacctcct 1680tcatggaaga ctacacttac cacttcgaac caggtaacga aatgatcttg ggttctcaca 1740tgttggaagt ttgtccaact gtcgctttgg accaaccaaa gatcgaagtt cacccattgt 1800ctatcggtgg taaagaagat ccagctagat tcgtcttcaa cggtatctct ggttccgcta 1860tccaagcctc tttggttgac atcggtggta gattcagatt ggttttgaac gaagtcaacg 1920gtcaagaaat cgaaaaggac atgccaaact tgccagttgc tagagtcttg tggaagccag 1980aaccatcttt gaagactgct gctgaagcct ggatcttggc tggtggtgct caccacacct 2040gtttgtctta cgaattgact gtcgaacaaa tgttggactg ggctgaaatg gctggtatcg 2100aatctgtttt gatctccaga gataccacta tccacaagtt gaagcacgaa ttgaagtgga 2160acgaagcctt gtacagattg caaaagtaat taattaatca tgtaattagt tatgtcacgc 2220ttacattcac gccctcctcc cacatccgct ctaaccgaaa aggaaggagt tagacaacct 2280gaagtctagg tccctattta ttttttttaa tagttatgtt agtattaaga acgttattta 2340tatttcaaat ttttcttttt tttctgtaca aacgcgtgta cgcatgtaac attatactga 2400aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcggg cggccgctct 2460agaactagta ccacaggtgt tgtcctctga ggacataaaa tacacaccga gattcatcaa 2520ctcattgctg gagttagcat atctacaatt gggtgaaatg gggagcgatt tgcaggcatt 2580tgctcggcat gccggtagag gtgtggtcaa taagagcgac ctcatgctat acctgagaaa 2640gcaacctgac ctacaggaaa gagttactca agaataagaa ttttcgtttt aaaacctaag 2700agtcacttta aaatttgtat acacttattt tttttataac ttatttaata ataaaaatca 2760taaatcataa gaaattcgct tactcttatt gaccgtagta agccttagca ccgtgctttc 2820tcaagtagtg cttgttcaac aaggtttgtt gcatgtctgg caattgtgga gccaattgtc 2880tacagaagat acccatgtaa gcaacttctt ccaagacgat agcgttgtga acagcatctt 2940cagcgttttt accccaagcg aatggaccgt gagagtgaac caagacacct ggcatttgag 3000cagcgtcgat accttgcttt tcgaaggttt caacgatgac gttaccagtt tcccattcgt 3060attcaccgtt gatttcagca tcggtcatct ttctagtaca tgggatagta ccgtagaagt 3120agtcagcgtg agtagtacca gtagctggga tagattgacc agcttgagcc cagatggtag 3180cgtgtctgga gtgagtgtgg acgataccac cgatagatgg gaaggcttgg tacaacaatc 3240tgtgagttgg ggtgtcggaa gatggcttct tagcaccttc aacgacttca ccggtttcga 3300tggaaacgac aaccatatcg tcagcagtca tgatggagta atcaacacca gatggcttga 3360taacgaagac acctctttct ctgtcaacag cagagacgtt accccaggtc aaggtgacca 3420agttgtgctt tggcaaagcc aagttagctt ccaaaacttg tctcttcaaa tcttccaaca 3480ttttgtttgt ttatgtgtgt ttattcgaaa ctaagttctt ggtgttttaa aactaaaaaa 3540aagactaact ataaaagtag aatttaagaa gtttaagaaa tagatttaca gaattacaat 3600caatacctac cgtctttata tacttattag tcaagtaggg gaataatttc agggaactgg 3660tttcaacctt ttttttcagc tttttccaaa tcagagagag cagaaggtaa tagaaggtgt 3720aagaaaatga gatagataca tgcgtgggtc aattgccttg tgtcatcatt tactccaggc 3780aggttgcatc actccattga ggttgtgccc gttttttgcc tgtttgtgcc cctgttctct 3840gtagttgcgc taagagaatg gacctatgaa ctgatggttg gtgaagaaaa caatattttg 3900gtgctgggat tctttttttt tctggatgcc agcttaaaaa gcgggctcca ttatatttag 3960tggatgccag gaataaactg ttcacccaga cacctacgat gttatatatt ctgtgtaacc 4020cgccccctat tttgggcatg tacgggttac agcagaatta aaaggctaat tttttgacta 4080aataaagtta ggaaaatcac tactattaat tatttacgta ttctttgaaa tggcagtatt 4140gataatgata aactcgaact agatctatcc gcggtgccgg cagatctatt taaatggcgc 4200gccgacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt 4260ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata 4320atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt 4380tgcggcattt

tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc 4440tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat 4500ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct 4560atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca 4620ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg 4680catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa 4740cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg 4800ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga 4860cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg 4920cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt 4980tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg 5040agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc 5100ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca 5160gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc 5220atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat 5280cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5340agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5400ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 5460accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct 5520tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5580cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg 5640gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5700gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5760gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 5820cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5880tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 5940ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg 6000ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat 6060taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc 6120agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc 6180gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa 6240cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc 6300ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 6360ccatgattac gccaagcttt ttctttccaa tttttttttt ttcgtcatta taaaaatcat 6420tacgaccgag attcccgggt aataactgat ataattaaat tgaagctcta atttgtgagt 6480ttagtataca tgcatttact tataatacag ttttttagtt ttgctggccg catcttctca 6540aatatgcttc ccagcctgct tttctgtaac gttcaccctc taccttagca tcccttccct 6600ttgcaaatag tcctcttcca acaataataa tgtcagatcc tgtagagacc acatcatcca 6660cggttctata ctgttgaccc aatgcgtctc ccttgtcatc taaacccaca ccgggtgtca 6720taatcaacca atcgtaacct tcatctcttc cacccatgtc tctttgagca ataaagccga 6780taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc cttagtatat tctccagtag 6840atagggagcc cttgcatgac aattctgcta acatcaaaag gcctctaggt tcctttgtta 6900cttcttctgc cgcctgcttc aaaccgctaa caatacctgg gcccaccaca ccgtgtgcat 6960tcgtaatgtc tgcccattct gctattctgt atacacccgc agagtactgc aatttgactg 7020tattaccaat gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac ttggcggata 7080atgcctttag cggcttaact gtgccctcca tggaaaaatc agtcaagata tccacatgtg 7140tttttagtaa acaaattttg ggacctaatg cttcaactaa ctccagtaat tccttggtgg 7200tacgaacatc caatgaagca cacaagtttg tttgcttttc gtgcatgata ttaaatagct 7260tggcagcaac aggactagga tgagtagcag cacgttcctt atatgtagct ttcgacatga 7320tttatcttcg tttcctgcag gtttttgttc tgtgcagttg ggttaagaat actgggcaat 7380ttcatgtttc ttcaacacta catatgcgta tatataccaa tctaagtctg tgctccttcc 7440ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa atttcaagga aaccgaaatc 7500aaaaaaaaga ataaaaaaaa aatgatgaat tgaaaagctt gcatgcctgc aggtcgactc 7560tagtatactc cgtctactgt acgatacact tccgctcagg tccttgtcct ttaacgaggc 7620cttaccactc ttttgttact ctattgatcc agctcagcaa aggcagtgtg atctaagatt 7680ctatcttcgc gatgtagtaa aactagctag accgagaaag agactagaaa tgcaaaaggc 7740acttctacaa tggctgccat cattattatc cgatgtgacg ctgcattttt tttttttttt 7800tttttttttt tttttttttt tttttttttt tttttttgta caaatatcat aaaaaaagag 7860aatcttttta agcaaggatt ttcttaactt cttcggcgac agcatcaccg acttcggtgg 7920tactgttgga accacctaaa tcaccagttc tgatacctgc atccaaaacc tttttaactg 7980catcttcaat ggctttacct tcttcaggca agttcaatga caatttcaac atcattgcag 8040cagacaagat agtggcgata gggttgacct tattctttgg caaatctgga gcggaaccat 8100ggcatggttc gtacaaacca aatgcggtgt tcttgtctgg caaagaggcc aaggacgcag 8160atggcaacaa acccaaggag cctgggataa cggaggcttc atcggagatg atatcaccaa 8220acatgttgct ggtgattata ataccattta ggtgggttgg gttcttaact aggatcatgg 8280cggcagaatc aatcaattga tgttgaactt tcaatgtagg gaattcgttc ttgatggttt 8340cctccacagt ttttctccat aatcttgaag aggccaaaac attagcttta tccaaggacc 8400aaataggcaa tggtggctca tgttgtaggg ccatgaaagc ggccattctt gtgattcttt 8460gcacttctgg aacggtgtat tgttcactat cccaagcgac accatcacca tcgtcttcct 8520ttctcttacc aaagtaaata cctcccacta attctctaac aacaacgaag tcagtacctt 8580tagcaaattg tggcttgatt ggagataagt ctaaaagaga gtcggatgca aagttacatg 8640gtcttaagtt ggcgtacaat tgaagttctt tacggatttt tagtaaacct tgttcaggtc 8700taacactacc ggtaccccat ttaggaccac ccacagcacc taacaaaacg gcatcagcct 8760tcttggaggc ttccagcgcc tcatctggaa gtggaacacc tgtagcatcg atagcagcac 8820caccaattaa atgattttcg aaatcgaact tgacattgga acgaacatca gaaatagctt 8880taagaacctt aatggcttcg gctgtgattt cttgaccaac gtggtcacct ggcaaaacga 8940cgatcttctt aggggcagac attacaatgg tatatccttg aaatatatat aaaaaaaaaa 9000aaaaaaaaaa aaaaaaaaaa tgcagcttct caatgatatt cgaatacgct ttgaggagat 9060acagcctaat atccgacaaa ctgttttaca gatttacgat cgtacttgtt acccatcatt 9120gaattttgaa catccgaacc tgggagtttt ccctgaaaca gatagtatat ttgaacctgt 9180ataataatat atagtctagc gctttacgga agacaatgta tgtatttcgg ttcctggaga 9240aactattgca tctattgcat aggtaatctt gcacgtcgca tccccggttc attttctgcg 9300tttccatctt gcacttcaat agcatatctt tgttaacgaa gcatctgtgc ttcattttgt 9360agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt 9420tacagaacag aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt 9480tttgtaaaac aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc 9540atttttacag aacagaaatg caacgcgaga gcgctatttt accaacaaag aatctatact 9600tcttttttgt tctacaaaaa tgcatcccga gagcgctatt tttctaacaa agcatcttag 9660attacttttt ttctcctttg tgcgctctat aatgcagtct cttgataact ttttgcactg 9720taggtccgtt aaggttagaa gaaggctact ttggtgtcta ttttctcttc cataaaaaaa 9780gcctgactcc acttcccgcg tttactgatt actagcgaag ctgcgggtgc attttttcaa 9840gataaaggca tccccgatta tattctatac cgatgtggat tgcgcatact ttgtgaacag 9900aaagtgatag cgttgatgat tcttcattgg tcagaaaatt atgaacggtt tcttctattt 9960tgtctctata tactacgtat aggaaatgtt tacattttcg tattgttttc gattcactct 10020atgaatagtt cttactacaa tttttttgtc taaagagtaa tactagagat aaacataaaa 10080aatgtagagg tcgagtttag atgcaagttc aaggagcgaa aggtggatgg gtaggttata 10140tagggatata gcacagagat atatagcaaa gagatacttt tgagcaatgt ttgtggaagc 10200ggtattcgca atattttagt agctcgttac agtccggtgc gtttttggtt ttttgaaagt 10260gcgtcttcag agcgcttttg gttttcaaaa gcgctctgaa gttcctatac tttctagaga 10320ataggaactt cggaatagga acttcaaagc gtttccgaaa acgagcgctt ccgaaaatgc 10380aacgcgagct gcgcacatac agctcactgt tcacgtcgca cctatatctg cgtgttgcct 10440gtatatatat atacatgaga agaacggcat agtgcgtgtt tatgcttaaa tgcgtactta 10500tatgcgtcta tttatgtagg atgaaaggta gtctagtacc tcctgtgata ttatcccatt 10560ccatgcgggg tatcgtatgc ttccttcagc actacccttt agctgttcta tatgctgcca 10620ctcctcaatt ggattagtct catccttcaa tgctatcatt tcctttgata ttggatcata 10680tgcatagtac cgagaaacta gaggatctcc cattaccgac atttgggcgc tatacgtgca 10740tatgttcatg tatgtatctg tatttaaaac acttttgtat tatttttcct catatatgtg 10800tataggttta tacggatgat ttaattatta cttcaccacc ctttatttca ggctgatatc 10860ttagccttgt tactagtcac cggtggc 1088760500PRTEscherichia coli 60Met Thr Ile Phe Asp Asn Tyr Glu Val Trp Phe Val Ile Gly Ser Gln1 5 10 15His Leu Tyr Gly Pro Glu Thr Leu Arg Gln Val Thr Gln His Ala Glu 20 25 30His Val Val Asn Ala Leu Asn Thr Glu Ala Lys Leu Pro Cys Lys Leu 35 40 45Val Leu Lys Pro Leu Gly Thr Thr Pro Asp Glu Ile Thr Ala Ile Cys 50 55 60Arg Asp Ala Asn Tyr Asp Asp Pro Cys Ala Gly Leu Val Val Trp Leu65 70 75 80His Thr Phe Ser Pro Ala Lys Met Trp Ile Asn Gly Leu Thr Met Leu 85 90 95Asn Lys Pro Leu Leu Gln Phe His Thr Gln Phe Asn Ala Ala Leu Pro 100 105 110Trp Asp Ser Ile Asp Met Asp Phe Met Asn Leu Asn Gln Thr Ala His 115 120 125Gly Gly Arg Glu Phe Gly Phe Ile Gly Ala Arg Met Arg Gln Gln His 130 135 140Ala Val Val Thr Gly His Trp Gln Asp Lys Gln Ala His Glu Arg Ile145 150 155 160Gly Ser Trp Met Arg Gln Ala Val Ser Lys Gln Asp Thr Arg His Leu 165 170 175Lys Val Cys Arg Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr Asp 180 185 190Gly Asp Lys Val Ala Ala Gln Ile Lys Phe Gly Phe Ser Val Asn Thr 195 200 205Trp Ala Val Gly Asp Leu Val Gln Val Val Asn Ser Ile Ser Asp Gly 210 215 220Asp Val Asn Ala Leu Val Asp Glu Tyr Glu Ser Cys Tyr Thr Met Thr225 230 235 240Pro Ala Thr Gln Ile His Gly Glu Lys Arg Gln Asn Val Leu Glu Ala 245 250 255Ala Arg Ile Glu Leu Gly Met Lys Arg Phe Leu Glu Gln Gly Gly Phe 260 265 270His Ala Phe Thr Thr Thr Phe Glu Asp Leu His Gly Leu Lys Gln Leu 275 280 285Pro Gly Leu Ala Val Gln Arg Leu Met Gln Gln Gly Tyr Gly Phe Ala 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Leu Arg Ile Met Lys Val305 310 315 320Met Ser Thr Gly Leu Gln Gly Gly Thr Ser Phe Met Glu Asp Tyr Thr 325 330 335Tyr His Phe Glu Lys Gly Asn Asp Leu Val Leu Gly Ser His Met Leu 340 345 350Glu Val Cys Pro Ser Ile Ala Val Glu Glu Lys Pro Ile Leu Asp Val 355 360 365Gln His Leu Gly Ile Gly Gly Lys Asp Asp Pro Ala Arg Leu Ile Phe 370 375 380Asn Thr Gln Thr Gly Pro Ala Ile Val Ala Ser Leu Ile Asp Leu Gly385 390 395 400Asp Arg Tyr Arg Leu Leu Val Asn Cys Ile Asp Thr Val Lys Thr Pro 405 410 415His Ser Leu Pro Lys Leu Pro Val Ala Asn Ala Leu Trp Lys Ala Gln 420 425 430Pro Asp Leu Pro Thr Ala Ser Glu Ala Trp Ile Leu Ala Gly Gly Ala 435 440 445His His Thr Val Phe Ser His Ala Leu Asn Leu Asn Asp Met Arg Gln 450 455 460Phe Ala Glu Met His Asp Ile Glu Ile Thr Val Ile Asp Asn Asp Thr465 470 475 480Arg Leu Pro Ala Phe Lys Asp Ala Leu Arg Trp Asn Glu Val Tyr Tyr 485 490 495Gly Phe Arg Arg 50061493PRTBacillus licheniformis 61Met Ile Gln Ala Lys Thr His Val Phe Trp Phe Val Thr Gly Ser Gln1 5 10 15His Leu Tyr Gly Glu Glu Ala Val Gln Glu Val Glu Glu His Ser Lys 20 25 30Met Ile Cys Asn Gly Leu Asn Asp Gly Asp Leu Arg Phe Gln Val Glu 35 40 45Tyr Lys Ala Val Ala Thr Ser Leu Asp Gly Val Arg Lys Leu Phe Glu 50 55 60Glu Ala Asn Arg Asp Glu Glu Cys Ala Gly Ile Ile Thr Trp Met His65 70 75 80Thr Phe Ser Pro Ala Lys Met Trp Ile Pro Gly Leu Ser Glu Leu Asn 85 90 95Lys Pro Leu Leu His Phe His Thr Gln Phe Asn Arg Asp Ile Pro Trp 100 105 110Asp Lys Ile Asp Met Asp Phe Met Asn Ile Asn Gln Ser Ala His Gly 115 120 125Asp Arg Glu Tyr Gly Phe Ile Gly Ala Arg Leu Gly Ile Pro Arg Lys 130 135 140Val Ile Ala Gly Tyr Trp Glu Asp Arg Glu Val Lys Arg Ser Ile Asp145 150 155 160Lys Trp Met Ser Ala Ala Val Ala Tyr Ile Glu Ser Arg His Ile Lys 165 170 175Val Ala Arg Phe Gly Asp Asn Met Arg Asn Val Ala Val Thr Glu Gly 180 185 190Asp Lys Ile Glu Ala Gln Ile Gln Leu Gly Trp Ser Val Asp Gly Tyr 195 200 205Gly Ile Gly Asp Leu Val Thr Glu Ile Asn Ala Val Ser Glu Gln Ser 210 215 220Leu Ser Glu Leu Ile Ser Glu Tyr Glu Glu Leu Tyr Glu Trp Pro Glu225 230 235 240Gly Glu Ala Ala Arg Glu Ser Val Lys Glu Gln Ala Arg Ile Glu Leu 245 250 255Gly Leu Lys Arg Phe Leu Ser Ser Gly Gly Tyr Thr Ala Phe Thr Thr 260 265 270Thr Phe Glu Asp Leu His Gly Met Lys Gln Leu Pro Gly Leu Ala Val 275 280 285Gln Arg Leu Met Ala Glu Gly Tyr Gly Phe Gly Gly Glu Gly Asp Trp 290 295 300Lys Thr Ala Ala Leu Val Arg Met Met Lys Met Met Ala Gly Gly Lys305 310 315 320Glu Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Phe Glu Pro Gly Asn 325 330 335Glu Met Ile Leu Gly Ser His Met Leu Glu Val Cys Pro Ser Ile Ala 340 345 350Glu His Lys Pro Arg Ile Glu Val His Pro Leu Ser Met Gly Ala Lys 355 360 365Asp Asp Pro Ala Arg Leu Val Phe Asp Gly Ile Ala Gly Pro Ala Val 370 375 380Asn Val Ser Leu Ile Asp Leu Gly Gly Arg Phe Arg Leu Val Ile Asn385 390 395 400Lys Val Glu Ala Val Lys Val Pro His Asp Met Pro Asn Leu Pro Val 405 410 415Ala Arg Val Leu Trp Lys Pro Gln Pro Ser Leu Arg Thr Ser Ala Glu 420 425 430Ala Trp Ile Leu Ala Gly Gly Ala His His Thr Cys Leu Ser Tyr Gln 435 440 445Leu Thr Ala Glu Gln Met Leu Asp Trp Ala Glu Met Ser Gly Ile Glu 450 455 460Ala Val Leu Ile Asn Arg Asp Thr Thr Ile Leu Asn Leu Arg Asn Glu465 470 475 480Leu Lys Trp Ser Glu Ala Ala Tyr Arg Leu Arg Lys Phe 485 49062488PRTClostridium acetobutylicum 62Met Leu Glu Asn Lys Lys Met Glu Phe Trp Phe Val Val Gly Ser Gln1 5 10 15His Leu Tyr Gly Glu Glu Ala Leu Lys Glu Val Arg Lys Asn Ser Glu 20 25 30Thr Ile Val Asp Glu Leu Asn Lys Ser Ala Asn Leu Pro Tyr Lys Ile 35 40 45Ile Phe Lys Asp Leu Ala Thr Ser Ala Asp Lys Ile Lys Glu Ile Met 50 55 60Lys Glu Val Asn Tyr Arg Asp Glu Val Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Met Trp Ile Ala Gly Thr Lys Ile Leu 85 90 95Gln Lys Pro Leu Leu His Phe Ala Thr Gln Tyr Asn Glu Asn Ile Pro 100 105 110Trp Lys Thr Ile Asp Met Asp Tyr Met Asn Leu His Gln Ser Ala His 115 120 125Gly Asp Arg Glu Tyr Gly Phe Ile Asn Ala Arg Leu Lys Lys His Asn 130 135 140Lys Val Val Val Gly Tyr Trp Lys Asp Lys Glu Val Gln Lys Gln Val145 150 155 160Ser Asp Trp Met Lys Val Ala Ala Gly Tyr Ile Ala Ser Glu Ser Ile 165 170 175Lys Val Ala Arg Phe Gly Asp Asn Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Gln Phe Gly Trp Thr Val Asp Tyr 195 200 205Phe Gly Ile Gly Asp Leu Val Ala Glu Met Asp Lys Val Ser Gln Asp 210 215 220Glu Ile Asn Lys Thr Tyr Glu Glu Phe Lys Asp Leu Tyr Ile Leu Asp225 230 235 240Pro Gly Glu Asn Asp Pro Ala Phe Tyr Glu Lys Gln Val Lys Glu Gln 245 250 255Ile Lys Ile Glu Ile Gly Leu Arg Arg Phe Leu Glu Lys Gly Asn Tyr 260 265 270Asn Ala Phe Thr Thr Asn Phe Glu Asp Leu Tyr Gly Met Lys Gln Leu 275 280 285Pro Gly Leu Ala Val Gln Arg Leu Asn Ala Glu Gly Tyr Gly Phe Ala 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Asp Arg Leu Leu Lys Val305 310 315 320Met Thr Asn Asn Thr Ala Thr Gly Phe Met Glu Asp Tyr Thr Tyr Glu 325 330 335Leu Ser Arg Gly Asn Glu Lys Ala Leu Gly Ala His Met Leu Glu Val 340 345 350Asp Pro Thr Phe Ala Ser Asp Lys Pro Lys Val Ile Val Lys Pro Leu 355 360 365Gly Ile Gly Asp Lys Glu Asp Pro Ala Arg Leu Ile Phe Asn Gly Ser 370 375 380Thr Gly Lys Gly Val Ala Val Ser Met Leu Asp Leu Gly Thr His Tyr385 390

395 400Arg Leu Ile Ile Asn Gly Leu Thr Ala Val Lys Pro Asp Glu Asp Met 405 410 415Pro Asn Leu Pro Val Ala Lys Met Val Trp Lys Pro Glu Pro Asn Phe 420 425 430Ile Glu Gly Val Lys Ser Trp Ile Tyr Ala Gly Gly Gly His His Thr 435 440 445Val Val Ser Leu Glu Leu Thr Val Glu Gln Val Tyr Asp Trp Ser Arg 450 455 460Met Val Gly Leu Glu Ala Val Ile Ile Asp Lys Asp Thr Lys Leu Arg465 470 475 480Asp Ile Ile Glu Lys Thr Thr Lys 48563474PRTLeuconostoc mesenteroides 63Met Ala Asp Ile Lys Asp Tyr Lys Phe Trp Phe Val Thr Gly Ser Gln1 5 10 15Phe Leu Tyr Gly Pro Glu Val Leu Lys Gln Val Glu Glu Asp Ser Lys 20 25 30Lys Ile Ile Glu Lys Leu Asn Glu Ser Gly Asn Leu Pro Tyr Pro Ile 35 40 45Glu Phe Lys Thr Val Gly Val Thr Ala Glu Asn Ile Thr Glu Ala Met 50 55 60Lys Glu Ala Asn Tyr Asp Asp Ser Val Ala Gly Val Ile Thr Trp Ala65 70 75 80His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Gln Leu Leu 85 90 95Asn Lys Pro Leu Leu His Leu Ala Thr Gln Met Leu Asn Asn Ile Pro 100 105 110Tyr Asp Ser Ile Asp Phe Asp Tyr Met Asn Leu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Tyr Ala Phe Ile Asn Ala Arg Leu Arg Leu Asn Asn 130 135 140Lys Ile Val Phe Gly His Trp Ala Asp Glu Ala Val Gln Val Gln Ile145 150 155 160Gly Lys Trp Met Asp Val Ala Val Ala Tyr Glu Glu Ser Phe Lys Ile 165 170 175Lys Val Val Thr Phe Ala Asp Lys Met Arg Asn Val Ala Val Thr Asp 180 185 190Gly Asp Lys Ile Glu Ala Gln Ile Lys Phe Gly Trp Thr Val Asp Tyr 195 200 205Trp Gly Val Gly Asp Leu Val Thr Tyr Val Asn Ala Ile Asp Asp Ala 210 215 220Asp Ile Asp Asn Leu Tyr Ile Glu Leu Gln Asp Lys Tyr Asp Phe Val225 230 235 240Ala Gly Gln Asn Asp Ser Glu Lys Tyr Glu His Asn Val Lys Tyr Gln 245 250 255Leu Arg Glu Tyr Leu Gly Ile Lys Arg Phe Leu Thr Asp Lys Gly Tyr 260 265 270Ser Ala Phe Thr Thr Asn Phe Glu Asp Leu Val Gly Leu Glu Gln Leu 275 280 285Pro Gly Leu Ala Ala Gln Leu Leu Met Ala Asp Gly Phe Gly Phe Ala 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Thr Arg Leu Leu Lys Ile305 310 315 320Val Ser His Asn Gln Ala Thr Ala Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335Leu Arg Gln Gly His Glu Ala Ile Leu Gly Ser His Met Leu Glu Val 340 345 350Asp Pro Thr Ile Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu 355 360 365Gly Ile Gly Gly Lys Glu Asp Pro Ala Arg Leu Val Phe Ser Gly Arg 370 375 380Thr Gly Asp Ala Val Asp Val Thr Ile Ser Asp Phe Gly Asp Glu Phe385 390 395 400Lys Leu Ile Ser Tyr Asp Val Thr Gly Asn Lys Pro Glu Ala Glu Thr 405 410 415Pro Tyr Leu Pro Val Ala Lys Gln Leu Trp Thr Pro Lys Ala Gly Leu 420 425 430Lys Ala Gly Ala Glu Gly Trp Leu Thr Val Gly Gly Gly His His Thr 435 440 445Thr Leu Ser Phe Ser Val Asp Ser Glu Gln Leu Thr Asp Leu Ala Asn 450 455 460Leu Phe Gly Val Thr Tyr Val Asp Ile Lys465 47064474PRTLactobacillus plantarum 64Met Leu Ser Val Pro Asp Tyr Glu Phe Trp Phe Val Thr Gly Ser Gln1 5 10 15His Leu Tyr Gly Glu Glu Gln Leu Lys Ser Val Ala Lys Asp Ala Gln 20 25 30Asp Ile Ala Asp Lys Leu Asn Ala Ser Gly Lys Leu Pro Tyr Lys Val 35 40 45Val Phe Lys Asp Val Met Thr Thr Ala Glu Ser Ile Thr Asn Phe Met 50 55 60Lys Glu Val Asn Tyr Asn Asp Lys Val Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Glu Leu Leu 85 90 95Gln Lys Pro Leu Leu His Leu Ala Thr Gln Tyr Leu Asn Asn Ile Pro 100 105 110Tyr Ala Asp Ile Asp Phe Asp Tyr Met Asn Leu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Tyr Ala Tyr Ile Asn Ala Arg Leu Gln Lys His Asn 130 135 140Lys Ile Val Tyr Gly Tyr Trp Gly Asp Glu Asp Val Gln Glu Gln Ile145 150 155 160Ala Arg Trp Glu Asp Val Ala Val Ala Tyr Asn Glu Ser Phe Lys Val 165 170 175Lys Val Ala Arg Phe Gly Asp Thr Met Arg Asn Val Ala Val Thr Glu 180 185 190Gly Asp Lys Val Glu Ala Gln Ile Lys Met Gly Trp Thr Val Asp Tyr 195 200 205Tyr Gly Ile Gly Asp Leu Val Glu Glu Ile Asn Lys Val Ser Asp Ala 210 215 220Asp Val Asp Lys Glu Tyr Ala Asp Leu Glu Ser Arg Tyr Glu Met Val225 230 235 240Gln Gly Asp Asn Asp Ala Asp Thr Tyr Lys His Ser Val Arg Val Gln 245 250 255Leu Ala Gln Tyr Leu Gly Ile Lys Arg Phe Leu Glu Arg Gly Gly Tyr 260 265 270Thr Ala Phe Thr Thr Asn Phe Glu Asp Leu Trp Gly Met Glu Gln Leu 275 280 285Pro Gly Leu Ala Ser Gln Leu Leu Ile Arg Asp Gly Tyr Gly Phe Gly 290 295 300Ala Glu Gly Asp Trp Lys Thr Ala Ala Leu Gly Arg Val Met Lys Ile305 310 315 320Met Ser His Asn Lys Gln Thr Ala Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335Leu Arg His Gly His Glu Ala Ile Leu Gly Ser His Met Leu Glu Val 340 345 350Asp Pro Ser Ile Ala Ser Asp Lys Pro Arg Val Glu Val His Pro Leu 355 360 365Asp Ile Gly Gly Lys Asp Asp Pro Ala Arg Leu Val Phe Thr Gly Ser 370 375 380Glu Gly Glu Ala Ile Asp Val Thr Val Ala Asp Phe Arg Asp Gly Phe385 390 395 400Lys Met Ile Ser Tyr Ala Val Asp Ala Asn Lys Pro Glu Ala Glu Thr 405 410 415Pro Asn Leu Pro Val Ala Lys Gln Leu Trp Thr Pro Lys Met Gly Leu 420 425 430Lys Lys Gly Ala Leu Glu Trp Met Gln Ala Gly Gly Gly His His Thr 435 440 445Met Leu Ser Phe Ser Leu Thr Glu Glu Gln Met Glu Asp Tyr Ala Thr 450 455 460Met Val Gly Met Thr Lys Ala Phe Leu Lys465 47065474PRTPediococcus pentosaceus 65Met Lys Lys Val Gln Asp Tyr Glu Phe Trp Phe Val Thr Gly Ser Gln1 5 10 15Phe Leu Tyr Gly Glu Glu Thr Leu Arg Ser Val Glu Lys Asp Ala Lys 20 25 30Glu Ile Val Asp Lys Leu Asn Glu Ser Lys Lys Leu Pro Tyr Pro Val 35 40 45Lys Phe Lys Leu Val Ala Thr Thr Ala Glu Asn Ile Thr Glu Val Met 50 55 60Lys Glu Val Asn Tyr Asn Asp Lys Val Ala Gly Val Ile Thr Trp Met65 70 75 80His Thr Phe Ser Pro Ala Lys Asn Trp Ile Arg Gly Thr Glu Leu Leu 85 90 95Gln Lys Pro Leu Leu His Leu Ala Thr Gln Phe Leu Asn His Ile Pro 100 105 110Tyr Asp Thr Ile Asp Phe Asp Tyr Met Asn Leu Asn Gln Ser Ala His 115 120 125Gly Asp Arg Glu Tyr Ala Phe Ile Asn Ala Arg Leu Arg Lys Asn Asn 130 135 140Lys Ile Ile Ser Gly Tyr Trp Gly Asp Glu Gly Ile Gln Lys Gln Ile145 150 155 160Ala Lys Trp Met Asp Val Ala Val Ala Tyr Asn Glu Ser Tyr Gly Ile 165 170 175Lys Val Val Thr Phe Ala Asp Lys Met Arg Asn Val Ala Val Thr Asp 180 185 190Gly Asp Lys Ile Glu Ala Gln Ile Lys Phe Gly Trp Thr Val Asp Tyr 195 200 205Trp Gly Val Ala Asp Leu Val Glu Glu Val Asn Ala Val Ser Asp Glu 210 215 220Asp Ile Asp Lys Lys Tyr Glu Glu Met Lys Asn Asp Tyr Asn Phe Val225 230 235 240Glu Gly Gln Asn Ser Ser Glu Lys Phe Glu His Asn Thr Lys Tyr Gln 245 250 255Ile Arg Glu Tyr Phe Gly Leu Lys Lys Phe Met Asp Asp Arg Gly Tyr 260 265 270Thr Ala Phe Thr Thr Asn Phe Glu Asp Leu Ala Gly Leu Glu Gln Leu 275 280 285Pro Gly Leu Ala Ala Gln Met Leu Met Ala Glu Gly Tyr Gly Phe Ala 290 295 300Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Asp Arg Leu Leu Lys Ile305 310 315 320Met Ala His Asn Lys Gln Thr Val Phe Met Glu Asp Tyr Thr Leu Asp 325 330 335Leu Arg Glu Gly His Glu Ala Ile Leu Gly Ser His Met Leu Glu Val 340 345 350Asp Pro Ser Ile Ala Ser Asp Thr Pro Arg Val Glu Val His Pro Leu 355 360 365Asp Ile Gly Gly Lys Glu Asp Pro Ala Arg Phe Val Phe Thr Gly Met 370 375 380Glu Gly Asp Ala Val Asp Val Thr Met Ala Asp Tyr Gly Asp Glu Phe385 390 395 400Lys Leu Met Ser Tyr Asp Val Thr Gly Asn Lys Thr Glu Lys Glu Thr 405 410 415Pro Tyr Leu Pro Val Ala Lys Gln Leu Trp Thr Pro Lys Gln Gly Trp 420 425 430Lys Gln Gly Ala Glu Gly Trp Leu Thr Leu Gly Gly Gly His His Thr 435 440 445Val Leu Ser Phe Ala Ile Asp Ala Glu Gln Leu Gln Asp Leu Ser Asn 450 455 460Met Phe Gly Leu Thr Tyr Val Asn Ile Lys465 4706633PRTartificial sequence237-269 amino acid motif with variation at multiple positionsVARIANT(2)..(2)R or KVARIANT(6)..(6)R or KVARIANT(11)..(11)I or MVARIANT(12)..(12)E or KVARIANT(14)..(14)I or MVARIANT(15)..(15)L or MVARIANT(16)..(16)V or T or DVARIANT(17)..(17)R or AVARIANT(18)..(18)E or NVARIANT(20)..(20)A or CVARIANT(21)..(21)K or N or RVARIANT(24)..(24)S or V or TVARIANT(28)..(28)E or QVARIANT(31)..(31)H or I or Y 66Ile Xaa Tyr Gln Ala Xaa Glu Glu Ile Ala Xaa Xaa Lys Xaa Xaa Xaa1 5 10 15Xaa Xaa Gly Xaa Xaa Ala Phe Xaa Asn Thr Phe Xaa Asp Leu Xaa Gly 20 25 30Met6733PRTartificial sequenceAmino acid sequence of the 237-269 Motif shown in FIG. 2AMISC_FEATURE(2)..(2)Arg or LysMISC_FEATURE(6)..(6)Arg or LysMISC_FEATURE(11)..(11)Ile or MetMISC_FEATURE(12)..(12)any amino acidMISC_FEATURE(14)..(14)Ile or MetMISC_FEATURE(15)..(15)Leu or MetMISC_FEATURE(16)..(16)any amino acidMISC_FEATURE(17)..(17)Arg or AlaMISC_FEATURE(18)..(18)Glu or AsnMISC_FEATURE(20)..(20)Ala or CysMISC_FEATURE(21)..(21)any amino acidMISC_FEATURE(24)..(24)any amino acidMISC_FEATURE(28)..(28)Gln or GluMISC_FEATURE(31)..(31)any amino acidMISC_FEATURE(32)..(32)any amino acid 67Ile Xaa Tyr Gln Ala Xaa Glu Glu Ile Ala Xaa Xaa Lys Xaa Xaa Xaa1 5 10 15Xaa Xaa Gly Xaa Xaa Ala Phe Xaa Asn Thr Phe Xaa Asp Leu Xaa Gly 20 25 30Met68477PRTRuminococcus flavefaciens 68Met Lys Phe Trp Phe Val Thr Gly Ser Gln Phe Leu Tyr Gly Glu Glu1 5 10 15Thr Leu Arg Gln Val Glu Glu Asp Ser Lys Lys Ile Val Asp Gly Leu 20 25 30Asp Leu Pro Phe Pro Val Glu Tyr Lys Met Thr Val Lys Lys Glu Ser 35 40 45Glu Ile Glu Arg Ile Ile Lys Glu Ala Asn Tyr Asp Asp Glu Cys Ala 50 55 60Gly Ile Ile Thr Phe Cys His Thr Phe Ser Pro Ser Lys Met Trp Ile65 70 75 80Asn Gly Leu Ala Leu Leu Gln Lys Pro Trp Leu His Phe His Thr Gln 85 90 95Phe Asn Glu Thr Ile Pro Asn Glu Gly Ile Asp Met Asp Tyr Met Asn 100 105 110Leu His Gln Ser Ala His Gly Asp Arg Glu His Gly Phe Ile Gly Ala 115 120 125Arg Leu Arg Met Pro Arg Ala Val Val Ala Gly His Trp Lys Asp Lys 130 135 140Lys Val Gln Glu Lys Ile Ala Glu Trp Gln Arg Ala Ala Val Gly Ala145 150 155 160Leu Phe Ser Lys Ser Leu Lys Ile Val Arg Phe Gly Asp Asn Met Arg 165 170 175Glu Val Ala Val Thr Glu Gly Asp Lys Ile Glu Ala Gln Leu Lys Leu 180 185 190Gly Trp Gln Val Asn Thr Phe Ala Val Gly Asp Leu Val Glu Ile Met 195 200 205Asn Ala Val Lys Asp Ala Glu Ile Asp Glu Leu Met Lys Glu Tyr Ala 210 215 220Glu Leu Tyr Asp Tyr Asp Lys Ala Asp Glu Glu Thr Ile Arg Tyr Gln225 230 235 240Ala Arg Glu Glu Ile Ala Ile Glu Lys Ile Leu Val Arg Glu Gly Ala 245 250 255Lys Ala Phe Ser Asn Thr Phe Glu Asp Leu His Gly Met Arg Gln Leu 260 265 270Pro Gly Leu Ala Thr Gln His Leu Met His Lys Gly Tyr Gly Phe Gly 275 280 285Ala Glu Gly Asp Trp Lys Thr Ala Gly Met Thr Ala Ile Val Lys Ala 290 295 300Met Tyr Pro Glu Gly Asn Thr Ser Phe Met Glu Asp Tyr Thr Tyr Asp305 310 315 320Tyr Lys His Glu Leu Ile Leu Gly Ser His Met Leu Glu Val Cys Pro 325 330 335Ser Ile Ala Ala Asp Lys Pro Arg Ile Glu Val His Lys Leu Gly Ile 340 345 350Gly Gly Lys Glu Ala Pro Ala Arg Ile Val Phe Glu Gly Arg Ala Gly 355 360 365Ser Ala Lys Ala Leu Ser Leu Ile Asp Ile Gly Gly Arg Phe Arg Leu 370 375 380Ile Ser Gln Asp Val Glu Cys Glu Lys Pro Phe Gln Ser Met Pro Asn385 390 395 400Leu Pro Val Ala Arg Thr Met Trp Lys Pro Ala Pro Ser Phe Leu Glu 405 410 415Gly Leu Glu Cys Trp Ile Ile Ala Gly Gly Ala His His Thr Val Leu 420 425 430Ser Tyr Asp Ile Thr Asp Glu Thr Val Arg Asp Phe Ala Arg Ile Met 435 440 445Gly Ile Glu Leu Val Val Ile Asn Lys Asp Thr Thr Lys Glu Lys Leu 450 455 460Glu Arg Asp Ile Met Ile Gly Asp Met Ile Tyr Gly Arg465 470 47569477PRTRuminococcus flavefaciens 69Met Lys Phe Trp Phe Ile Thr Gly Ser Gln Phe Leu Tyr Gly Glu Glu1 5 10 15Thr Leu Arg Gln Val Asp Glu Asp Ser Lys Lys Ile Val Ala Gly Leu 20 25 30Lys Leu Pro Phe Pro Val Glu Tyr Lys Ser Thr Val Lys Thr Glu Ser 35 40 45Glu Ile Gln Arg Ile Ile Lys Glu Ala Asn Phe Asp Asp Glu Cys Ala 50 55 60Gly Val Ile Thr Phe Cys His Thr Phe Ser Pro Ser Lys Met Trp Ile65 70 75 80Asn Gly Leu Ala Leu Leu Gln Lys Pro Trp Leu His Phe His Thr Gln 85 90 95Phe Asn Glu Thr Ile Pro Asn Glu Ala Ile Asp Met Asp Tyr Met Asn 100 105 110Leu His Gln Ser Ala His Gly Asp Arg Glu His Gly Phe Ile Gly Ala 115 120 125Arg Leu Arg Met Pro Arg Ala Val Val Ala Gly His Trp Gln Asp Pro 130 135 140Glu Val Gln Ala Lys Ile Ala Glu Trp Gln Arg Ala Ala Val Gly Val145 150 155 160Met Phe Ser Lys Ser Leu Lys Ile Val Arg Phe Gly Asp Asn Met Arg 165 170 175Glu Val Ala Val Thr Glu Gly Asp Lys Ile Glu Ala Gln Leu Lys Leu 180 185 190Gly Trp Gln Val Asn Thr Phe Ala Val Gly Asp Leu Val Glu Ile Met 195 200 205Asn Ala Val Thr Asp Ala Glu Ile Asp Ala Leu Met Lys Glu Tyr Ala 210 215 220Glu Leu Tyr Asp Tyr Lys Lys Glu Asp Glu Glu Thr Ile Arg Tyr Gln225 230 235 240Ala Arg Glu Glu Ile Ala Ile Glu Lys Ile Leu Val Arg Glu Gly Ala 245 250 255Lys Ala Tyr Ser Asn Thr Phe Glu Asp Leu

His Gly Met Lys Gln Leu 260 265 270Pro Gly Leu Ala Thr Gln His Leu Met His Lys Gly Tyr Gly Phe Gly 275 280 285Ala Glu Gly Asp Trp Lys Thr Ala Gly Met Thr Ala Ile Val Lys Ala 290 295 300Met Tyr Pro Glu Gly Asn Thr Ser Phe Met Glu Asp Tyr Thr Tyr Asp305 310 315 320Tyr Lys Gln Glu Leu Ile Leu Gly Ser His Met Leu Glu Val Cys Pro 325 330 335Ser Ile Ala Ala Asp Arg Pro Arg Ile Glu Val His Lys Leu Gly Ile 340 345 350Gly Gly Lys Glu Pro Pro Ala Arg Ile Val Phe Glu Gly Lys Ala Gly 355 360 365Ser Ala Lys Val Leu Ser Leu Ile Asp Ile Gly Gly Arg Leu Arg Leu 370 375 380Ile Gln Gln Asp Ile Glu Cys Val Lys Pro Phe Gln Ser Met Pro Asn385 390 395 400Leu Pro Val Ala Arg Thr Met Trp Arg Pro Ala Pro Ser Phe Leu Asp 405 410 415Gly Leu Glu Cys Trp Ile Ile Ala Gly Gly Ala His His Thr Val Leu 420 425 430Ser Tyr Asp Ile Ser Asp Glu Ala Val Arg Ser Phe Ala Arg Ile Met 435 440 445Gly Ile Glu Leu Val Val Ile Asn Lys Asp Thr Thr Val Asn Gly Leu 450 455 460Glu Arg Asp Ile Met Ile Gly Asp Val Ile Tyr Gly Arg465 470 47570477PRTRuminococcus flavefaciens 70Met Lys Phe Trp Phe Ile Thr Gly Ser Gln Phe Leu Tyr Gly Glu Glu1 5 10 15Thr Leu Arg Gln Val Asp Glu Asp Ser Lys Lys Ile Val Ala Gly Leu 20 25 30Lys Leu Pro Phe Pro Val Glu Tyr Lys Ser Thr Val Lys Thr Glu Arg 35 40 45Glu Ile Glu Arg Ile Ile Lys Glu Ala Asn Tyr Asp Asp Glu Cys Ala 50 55 60Gly Ile Ile Thr Phe Cys His Thr Phe Ser Pro Ser Lys Met Trp Ile65 70 75 80Asn Gly Leu Ala Leu Leu Gln Lys Pro Trp Leu His Phe His Thr Gln 85 90 95Phe Asn Glu Thr Ile Pro Asn Glu Ala Ile Asp Met Asp Tyr Met Asn 100 105 110Leu His Gln Ser Ala His Gly Asp Arg Glu His Gly Phe Ile Gly Ala 115 120 125Arg Leu Arg Met Pro Arg Ala Val Val Ala Gly His Trp Gln Asp Pro 130 135 140Glu Val Gln Ala Lys Ile Ala Glu Trp Gln Arg Ala Ala Val Gly Val145 150 155 160Met Phe Ser Lys Ser Leu Lys Ile Val Arg Phe Gly Asp Asn Met Arg 165 170 175Glu Val Ala Val Thr Glu Gly Asp Lys Val Glu Ala Gln Leu Lys Leu 180 185 190Gly Trp Gln Val Asn Thr Phe Ala Val Gly Asp Leu Val Glu Ile Met 195 200 205Asn Ala Val Thr Asn Thr Glu Ile Asp Ala Leu Met Lys Glu Tyr Ala 210 215 220Glu Leu Tyr Asp Tyr Lys Lys Glu Asp Glu Glu Thr Ile Arg Tyr Gln225 230 235 240Ala Arg Glu Glu Ile Ala Ile Glu Lys Ile Leu Val Arg Glu Gly Ala 245 250 255Lys Ala Phe Ser Asn Thr Phe Glu Asp Leu His Gly Met Lys Gln Leu 260 265 270Pro Gly Leu Ala Thr Gln His Leu Met His Lys Gly Tyr Gly Phe Gly 275 280 285Ala Glu Gly Asp Trp Lys Thr Ala Gly Met Thr Ala Ile Val Lys Ala 290 295 300Met Tyr Pro Asp Gly Asn Thr Ser Phe Met Glu Asp Tyr Thr Tyr Asp305 310 315 320Tyr Lys Gln Gln Leu Ile Leu Gly Ser His Met Leu Glu Val Cys Pro 325 330 335Ser Ile Ala Ala Asp Lys Pro Arg Ile Glu Val His Lys Leu Gly Ile 340 345 350Gly Gly Lys Glu Pro Pro Ala Arg Ile Val Phe Glu Gly Lys Ala Gly 355 360 365Ser Ala Lys Ala Leu Ser Leu Ile Asp Ile Gly Gly Arg Leu Arg Leu 370 375 380Ile Ser Gln Asp Val Glu Cys Val Lys Pro Phe Gln Ser Met Pro Asn385 390 395 400Leu Pro Val Ala Arg Thr Met Trp Arg Pro Ala Pro Ser Phe Leu Glu 405 410 415Gly Leu Glu Cys Trp Ile Val Ala Gly Gly Ala His His Thr Val Leu 420 425 430Ser Tyr Asp Ile Ser Asp Glu Ala Val Arg Ser Phe Ala Arg Ile Met 435 440 445Gly Ile Glu Leu Val Val Ile Asn Lys Asp Thr Thr Val Asn Gly Leu 450 455 460Glu Arg Asp Ile Met Ile Gly Asp Val Ile Tyr Gly Arg465 470 47571477PRTRuminococcus flavefaciens 71Met Lys Phe Trp Phe Val Thr Gly Ser Gln Phe Leu Tyr Gly Glu Glu1 5 10 15Thr Leu Arg Gln Val Glu Glu Asp Ser Lys Lys Ile Val Ala Gly Leu 20 25 30Lys Leu Pro Phe Pro Val Glu Tyr Lys Leu Thr Val Lys Lys Glu Ala 35 40 45Glu Ile Thr Lys Ile Ile Lys Glu Ala Asn Tyr Asp Asp Glu Cys Ala 50 55 60Gly Ile Ile Thr Phe Cys His Thr Phe Ser Pro Ser Lys Met Trp Ile65 70 75 80Asn Gly Leu Arg Ser Leu Gln Lys Pro Trp Leu His Phe His Thr Gln 85 90 95Phe Asn Asp Asn Ile Pro Asn Asp Ala Ile Asp Met Asp Tyr Met Asn 100 105 110Leu His Gln Ser Ala His Gly Asp Arg Glu His Gly Phe Ile Gly Ala 115 120 125Arg Leu Arg Met Pro Arg Ala Val Val Ala Gly His Trp Ala Asp Pro 130 135 140Ala Val Gln Glu Lys Ile Ala Asp Trp Met Arg Ala Ala Val Gly Val145 150 155 160Gln Phe Ser Lys Ser Leu Lys Ile Val Arg Phe Gly Asp Asn Met Arg 165 170 175Glu Val Ala Val Thr Glu Gly Asp Lys Ile Glu Ala Gln Ile Lys Leu 180 185 190Gly Trp Gln Val Asn Thr Phe Ala Val Gly Asp Leu Val Gln Ile Met 195 200 205Asn Ala Val Thr Asp Ala Glu Ile Asp Ala Leu Met Lys Glu Tyr Ala 210 215 220Glu Leu Tyr Asp Phe Asp Lys Ala Asp Glu Glu Cys Ile Arg Tyr Gln225 230 235 240Ala Arg Glu Glu Ile Ala Ile Glu Lys Ile Leu Val Arg Glu Gly Ala 245 250 255Met Ala Phe Ser Asn Thr Phe Glu Asp Leu His Gly Met Lys Gln Leu 260 265 270Pro Gly Leu Ala Thr Gln His Leu Met His Lys Gly Tyr Gly Phe Gly 275 280 285Ala Glu Gly Asp Trp Lys Thr Ala Gly Met Thr Ala Ile Ile Lys Ala 290 295 300Met Tyr Pro Asp Gly Asn Thr Ser Phe Met Glu Asp Tyr Asn Tyr Asp305 310 315 320Tyr Lys His Glu Leu Ile Leu Gly Ala His Met Leu Glu Val Cys Pro 325 330 335Ser Ile Ala Ala Gly Arg Pro Arg Ile Glu Val His Pro Leu Gly Ile 340 345 350Gly Gly Lys Asp Ala Pro Ala Arg Ile Val Phe Glu Gly Lys Ala Gly 355 360 365Ser Ala Lys Ala Ile Ser Leu Ile Asp Ile Gly Gly Arg Leu Arg Leu 370 375 380Ile Ala Gln Asp Val Glu Cys Glu Lys Pro Phe Gln Thr Met Pro Asn385 390 395 400Leu Pro Val Ala Arg Thr Met Trp Lys Pro Ala Pro Ser Phe Leu Glu 405 410 415Gly Leu Glu Cys Trp Ile Ile Ala Gly Gly Ala His His Thr Val Leu 420 425 430Ser Tyr Asp Ile Ser Asp Glu Thr Val Arg Asp Phe Ala Arg Ile Met 435 440 445Gly Ile Glu Leu Val Val Ile Asn Lys Asn Thr Asn Lys Tyr Gln Leu 450 455 460Glu Arg Asp Met Met Ile Gly Asp Val Ile Tyr Gly Arg465 470 475

Claims

1. A recombinant yeast cell comprising an arabinose utilization pathway that comprises a polypeptide having arabinose isomerase activity, wherein the yeast cell comprises a heterologous polynucleotide encoding said polypeptide, wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:67, to and wherein the position of the motif in the polypeptide corresponds with positions 237-269 of SEQ ID NO:7.

2. The recombinant yeast cell of claim 1, wherein the motif comprises at least seventeen amino acids selected from the group consisting of: (a) I at position 237; (b) R or K at position 238; (c) Y at position 239; (d) R or K at position 242; (e) E at position 243; (f) I at position 245; (g) A at position 246; (h) I or M at position 247; (i) K at position 249; (j) I or M at position 250; (k) R or A at position 253; (l) E or N at position 254; (m) G at position 255; (n) A or C at position 256; (o) F at position 259; (p) N at position 261; (q) T at position 262; (r) Q or E at position 264; and (s) M at position 269.

3. The recombinant yeast cell of claim 1, wherein the polypeptide comprises a motif that is SEQ ID NO:67.

4. The recombinant yeast cell of claim 1, wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:66.

5. The recombinant yeast cell of claim 4, wherein the polypeptide comprises a motif that is SEQ ID NO:66.

6. The recombinant yeast cell of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:7, 8, 10, 17, 18, or 19.

7. The recombinant yeast cell of claim 1, further comprising a metabolic pathway that produces a target compound, optionally wherein the target compound is ethanol, butanol, or 1,3-propanediol.

8. A method for producing a yeast cell having arabinose isomerase activity, said method comprising: (a) providing a yeast cell lacking arabinose isomerase; and (b) introducing a heterologous polynucleotide into the yeast cell, wherein the heterologous polynucleotide encodes a polypeptide having arabinose isomerase activity, and wherein the polypeptide comprises a motif that is at least 90% identical to SEQ ID NO:67.

9. The method of claim 8, wherein: (i) the yeast cell of step (a) comprises one or more polynucleotides encoding enzymes, except an arabinose isomerase, of an arabinose utilization pathway, or (ii) step (b) further comprises introducing, into the yeast cell, one or more polynucleotides encoding enzymes of an arabinose utilization pathway, wherein this further introduction is at the same time of, or after, said introducing the heterologous polynucleotide encoding the polypeptide having arabinose isomerase activity.

10. A method of producing a target compound from arabinose comprising: (a) providing the recombinant yeast cell of claim 7; (b) growing the yeast cell of (a) in medium comprising arabinose, wherein the target compound is produced; and c) optionally isolating the target compound of (b).

11. The method of claim 10, wherein the target compound is ethanol, butanol, or 1,3-propanediol.

12. A recombinant yeast cell comprising an arabinose utilization pathway that comprises a polypeptide having arabinose isomerase activity, wherein the yeast cell comprises a heterologous polynucleotide encoding said polypeptide, and wherein the polypeptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:15 or 20.

13. A method of producing a target compound from arabinose comprising: (a) providing the recombinant yeast cell of claim 12; (b) growing the yeast cell of (a) in medium comprising arabinose, wherein the target compound is produced; and c) optionally isolating the target compound of (b).